CELL LINES EXPRESSING CFTR AND METHODS OF USING THEM

Description

This application claims the benefit of U.S. Provisional Application 61/149,312, filed Feb. 2, 2009, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 16, 2010, is named 002298WO.txt and is 40,540 bytes in size.

FIELD OF THE INVENTION

The invention relates to cystic fibrosis transmembrane conductance regulator (CFTR) and cells and cell lines stably expressing CFTR. The invention further provides methods of making such cells and cell lines. The CFTR-expressing cells and cell lines provided herein are useful in identifying modulators of CFTR.

BACKGROUND

Cystic fibrosis is the most common genetic disease in the United States, and is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is a transmembrane ion channel protein that transports chloride ions and other anions. The chloride channels are present in the apical plasma membranes of epithelial cells in the lung, sweat glands, pancreas, and other tissues. CFTR regulates ion flux and helps control the movement of water in sales and maintain the fluidity of mucus and other secretions. Chloride transport is induced by an increase in cyclic adenosine monophosphate (cAMP), which activates protein kinase A to phosphorylate the channel on the regulatory “R” domain.

CFTR is a member of the ABC transporter family. It contains two ATP-binding cassettes. ATP binding, hydrolysis and cAMP-dependent phosphorylation are required for channel opening. CFTR is encoded by a single large gene consisting of 24 exons. CFTR ion channel function is associated with a wide range of disorders, including cystic fibrosis, congenital absence of the vas deferens, secretory diarrhea, and emphysema. To date, more than 1000 distinct mutations have been identified in CFTR. The most common CFTR mutation is deletion of phenylalanine at residue 508 (ΔF508) in its amino acid sequence. This mutation is present in approximately 70% of cystic fibrosis patients.

The discovery of new and improved therapeutics that specifically target CFTR has been hampered by the lack of robust, physiologically relevant cell-based systems that are amenable to high-throughput formats for identifying and testing CFTR modulators, particularly high-throughput formats that allow various members of the CFTR family of mutants to be compared. Cell-based systems are preferred for drug discovery and validation because they provide a functional assay for a compound as opposed to cell-free systems, which only provide a binding assay. Moreover, cell-based systems have the advantage of simultaneously testing cytotoxicity. Ideally, cell-based systems should also stably express the target protein. It is also desirable for a cell-based system to be reproducible. The present invention addresses these problems.

SUMMARY OF THE INVENTION

We have discovered new and useful cells and cell lines and collections of cell lines that express various forms of CFTR. These cells, cell lines, and collections thereof are useful in cell-based assays, in particular high-throughput assays to study the fucntions of CFTR and to screen for CFTR modulators.

Accordingly, the invention provides a cell or cell line engineered to stably express CFTR, e.g., a functional CFTR or a mutant (e.g., dysfunctional) CFTR. In some embodiments, the CFTR is expressed in a cell from an introduced nucleic acid encoding it. In some embodiments, the CFTR is expressed in a cell from an endogenous nucleic acid activated by engineered gene activation.

The cells or cell lines of the invention may be eukaryotic c cells (e.g., mammalian cells), and optionally do not express CFTR endogenously (or in the case of gene activation, do not express CFTR endogenously prior to gene activation). The cells may be primary or immortalized cells, may be cells of, for example, primate (e.g., human or monkey), rodent (e.g., mouse, rat, or hamster), or insect (e.g., fruit fly) origin. In some embodiments, the cells are capable of forming polarized monolayers. The CFTR expressed in the cells or cell lines of the invention may be mammalian, such as rat, mouse, rabbit, goat, dog, cow, pig, or primate (e.g., human).

In some embodiments, the cells and cell lines of the invention have a Z′ factor of at least 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8 or 0.85 in an assay, for example, a high throughput cell-based assay. In some embodiments, the cells or cell lines of the invention are maintained in the absence of selective pressure, e.g., antibiotics. In some embodiments, the CFTR expressed by the cells or cell lines does not comprise any polypeptide tag. In some embodiments, the cells or cell lines do not express any other introduced protein, including auto-fluorescent proteins (e.g., yellow fluorescent protein (YFP) or variants thereof).

In some embodiments, the cells or cell lines of the invention stably express CFTR at a consistent level in the absence of selective pressure for at least 15 days, 30 days, 45 days, 60 days, 75 days, 100 days, 120 days, or 150 days.

In another aspect of the invention, the cells or cell lines express a human CFTR. The CFTR may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2; a polypeptide t least 95% sequence identity to SEQ ID NO: 2; a polypeptide encoded by a nucleic acid that hybridizes to SEQ ID NO: 1 under stringent conditions; or a polypeptide that is arm allelic variant of SEQ ID NO: 2. The CFTR may also be encoded by a nucleic acid having the sequence set forth in SEQ ID NO: 1; a nucleic acid that hybridizes to SEQ ID NO: 1 under stringent conditions; a nucleic acid that encodes the polypeptide of SEQ ID NO: 2; a nucleic acid with at least 95% sequence identity to SEQ. ID NO: 1; or a nucleic acid that is an allelic variant of SEQ ID NO: 1. The CFTR may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7 or a polypeptide encoded by a nucleic acid sequence set forth in SEQ ID NO: 4.

In another aspect, the invention provides a collection of the cells or cells lines that express different forms (i.e., mutant forms) of CFTR. In some embodiments, the cells or cell lines in the collection comprise at least 2, at least 5, at least 10, at least 15, or at least 20 different cells or cell lines, each expressing at least a different form (i.e., mutant form) of CFTR. In some embodiments, the cells or cell lines in the collection are matched to share physiological properties (e.g., cell type, metabolism, cell passage (age), growth rate, adherence to a tissue culture surface, Z′ factor, expression level of CFTR) to allow parallel processing and accurate assay readouts. These can be achieved by generating and growing the cells and cell lines under identical conditions, achievable by, e.g., automation. In some embodiments, the Z′ factor is determined in the absence of a protein trafficking corrector. A protein trafficking corrector is a substance that aids maturation of improperly folded CFTR mutant by directly or indirectly interacting with the mutant CFTR at its transmembrane level and facilitates the mutant CFTR to reach the cell membrane.

In another aspect, the invention provides a method for producing the cells or cell lines of the invention, comprising the steps of: (a) introducing a vector comprising a nucleic acid encoding CFTR (e.g., human CFTR) into a host cell; or introducing one or more nucleic acid sequences that activate expression of endogenous CFTR (e.g., human CFTR); (b) introducing a molecular beacon or fluorogenic probe that detects the expression of CFTR into the host cell produced in step (a); and (c) isolating a cell that expresses CFTR. In some embodiments, the method comprises the additional step of generating a cell line from the cell isolated in step (c). The host cells may be eukaryotic cells such as mammalian cells, and may optionally do not express CFTR endogenously.

In some embodiments, the method of producing cells and cell lines of the invention utilizes a fluorescence activated cell sorter to isolate a cell that expresses CFTR. In some embodiments, the cell or cell lines of the collection are produced in parallel.

In another aspect, the invention provides a method for identifying a modulator of a CFTR function, comprising the steps of exposing a cell or cell line of the invention or a collection of the cell lines to a test compound; and detecting in a cell a change in a CFTR function, wherein a change indicates that the test compound is a CFTR modulator. In some embodiments, the detecting step can be a membrane potential assay, a yellow fluorescent protein (YFP) quench assay, an electrophysiology assay, a binding assay, or an Ussing chamber assay. In some embodiments, the assay in the detecting step is performed in the absence of a protein trafficking corrector. Test compounds used in the method may include a small molecule, a chemical moiety, a polypeptide, or an antibody. In other embodiments, the test compound may be a library of compounds. The library may be a small molecule library, a combinatorial library, a peptide library, or an antibody library.

In a further aspect, the invention provides a cell engineered to stably express CFTR at a consistent level over time. The cell may be made by a method comprising the steps of a) providing a plurality of cells that express mRNA(s) encoding the CFTR; b) dispersing the cells individually into individual culture vessels, thereby providing a plurality of separate cell cultures; c) culturing the cells under a set of desired culture conditions using automated cell culture methods characterized in that the conditions are substantially identical for each of the separate cell cultures, during which culturing the number of cells per separate cell culture is normalized, and wherein the separate cultures are passaged on the same schedule; d) assaying the separate cell cultures to measure expression of the CFTR at least twice; and e) identifying a separate cell culture that expresses the CFTR at a consistent level in both assays, thereby obtaining said cell.

In another aspect, the invention provides a method for isolating a cell that endogenously expresses CFTR, comprising the steps of: a) providing a population of cells; b) introducing into the cells a molecular beacon that detects expression of CFTR; and c) isolating cells that express CFTR. In some embodiments, the population of cells comprises cells that do not endogenously express CFTR. In some embodiments, the isolated cells that express CFTR prior to said isolating are not known to express CFTR. In some embodiments, the method further comprises, prior to said isolating step c), the step of increasing genetic variability.

In another aspect, the invention provides a use of a composition comprising a compound of the formula:

to increase the expression level of a CFTR on the cell plasma membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show that stable CFTR-expressing cell lines produced exhibit significantly enhanced and robust CFTR surface expression. Ion-flux in response to activated CFTR expression was measured by a high-throughput compatible fluorescence membrane potential assay. FIG. 1A compares stable CFTR-expressing cell line 1 to transiently CFTR-transfected cells and control cells lacking CFTR. FIG. 1B compares stable CFTR-expressing cell line 1 (from FIG. 1A) to other stable CFTR-expressing clones produced (M11, J5, E15, and O1).

FIG. 2 displays dose response curves from a high-throughput compatible fluorescence membrane potential assay of CFTR. The assay measured the response of produced stable CFTR-expressing cell lines to forskolin, an agonist of CFTR. The EC₅₀ value for forskolin in the tested cell lines as 256 nM. A Z′ value of at least 0.82 was obtained for the high-throughput compatible fluorescence membrane potential assay.

FIGS. 3A-3F show that stable CFTR-ΔF508 expressing CHO cell clones can be identified from non-responding clones from a population of CHO cells. Stable CFTR-ΔF508 expressing clones were able to rescue cell surface expression of CFTR-ΔF508 from entrapment in intracellular compartments, in the presence or absence of a protein trafficking corrector—Chembridge compound #5932794a (San Diego, Calif.). This compound is N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide, and has the formula of

Non-responding clones were not able to rescue cell surface expression of CFTR-ΔF508 from entrapment in intracellular compartments, either in the presence or absence of the protein trafficking corrector. Ion-flux in response to activated CFTR-ΔF508 expression was measured by a high-throughput compatible fluorescence membrane potential assay. FIG. 3A shows pharmacological response of a stable CFTR-ΔF508 expressing clone in the presence of a blue membrane potential dye and the protein trafficking corrector (15-25 μM) when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace). FIG. 3B shows pharmacological response of a non-responding clone in the presence of a blue membrane potential dye and the protein trafficking corrector (15-25 μM, same as in 3A) when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace). FIG. 3C shows pharmacological response of a stable CFTR-ΔF508 expressing clone in the presence of an AnaSpec membrane potential dye and the protein trafficking corrector (15-25 μM, same as in 3A, 3B) when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace). FIG. 3D shows pharmacological response of a non-responding clone in the presence of an AnaSpec membrane potential dye and the protein trafficking corrector (15-25 μM, same as in 3A, 3B, 3C) when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace). FIG. 3E shows pharmacological response of a stable CFTR-ΔF508 expressing clone in the presence of an AnaSpec membrane potential dye and without the protein trafficking corrector when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace). FIG. 3F shows pharmacological response of a non-responding clone in the presence of an AnaSpec membrane potential dye and without the protein trafficking corrector when challenged either by an agonist cocktail of forskolin (30 μM)+IBMX (100 μM) (black trace) or DMSO+Buffer (grey trace).

DETAILED DISCLOSURE

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The materials, methods, and examples are illustrative only and not intended to be limiting.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term “stable” or “stably expressing” is meant to distinguish the cells and cell lines of the invention from cells with transient expression as the terms “stable expression” and “transient expression” would be understood by a person of skill in the art.

The term “cell line” or “clonal cell line” refers to a population of cells that are all progeny of a single original cell. As used herein, cell lines are maintained in vitro in cell culture and may be frozen in aliquots to establish banks of clonal cells.

The term “stringent conditions” or “stringent hybridization conditions” describe temperature and salt conditions for hybridizing one or more nucleic acid probes to a nucleic acid sample and washing off probes that have not bound specifically to target nucleic acids in the sample. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. An example of stringent hybridization conditions is hybridization in 6×SSC at about 45° C., followed by at least one wash in 0.2×SSC, 0.1% SDS at 60° C. A further example of stringent hybridization conditions is hybridization in 6×SSC at about 45° C., followed by at least one wash in 0.2×SSC, 0.1% SDS at 65° C. Stringent conditions include hybridization in 0.5M sodium phosphate, 7% SDS at 65° C., followed by at least one wash at 0.2×SSC, 1% SDS at 65° C.

The phrase “percent identical” or “percent identity” in connection with amino acid and/or nucleic acid sequences refers to the similarity between at least two different sequences. This percent identity can be determined by standard alignment algorithms, for example, the Basic Local Alignment Tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol., 215: 403-410); the algorithm of Needleman et al. ((1970) J. Mol. Biol., 48: 444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11-17). A set of parameters may be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) that has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity is usually calculated by comparing sequences of similar length. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, the GCG Wisconsin Package (Accelrys, Inc.) contains programs such as “Gap” and “Bestfit” that can be used with default parameters to determine sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutant thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters. A program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). The length of polypeptide sequences compared for identity will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. The length of a DNA sequence compared for identity will generally be at least about 48 nucleic acid residues, usually at least about 60 nucleic acid residues, more usually at least about 72 nucleic acid residues, typically at least about 84 nucleic acid residues, and preferably more than about 105 nucleic acid residues.

The phrase “substantially as set out,” “substantially identical” or “substantially homologous” in connection with an amino acid nucleotide sequence means that the relevant amino acid or nucleotide sequence will be identical to or have differences (through conserved amino acid substitutions) in comparison to the sequences that are set out. Insubstantial differences include minor amino acid changes, such as 1 or 2 substitutions in a 50 amino acid sequence of a specified region. Insubstantial differences may have deleterious effect.

The terms “potentiator”, “corrector”, “agonist” or “activator” refer to a compound or substance that activates a biological function of CFTR, e.g., increases ion conductance via CFTR. As used herein, a potentiator, corrector or activator may act upon a CFTR or upon a specific subset of different forms (e.g., mutant forms) of CFTR.

The terms “inhibitor”, “antagonist” or “blocker” refers to a compound or substance that decreases a biological function of CFTR, e.g., decreases ion conductance via CFTR. As used herein, an inhibitor or blocker may act upon a CFTR or upon a specific subset of different forms (e.g., mutant forms) of CFTR.

The term “modulator” refers to a compound or substance that alters a structure, conformation, biochemical or biophysical property or functionality of a CFTR either positively or negatively. The modulator can be a CFTR agonist (potentiator, corrector, or activator) or antagonist (inhibitor or blocker), including partial agonists or antagonists, selective agonists or antagonists and inverse agonists, and can be an allosteric modulator. A substance or compound is a modulator even if its modulating activity changes under different conditions or concentrations or with respect to different forms (e.g., mutant forms) of CFTR. As used herein, a modulator may affect the ion conductance of a CFTR, the response of a CFTR to another regulatory compound, or the selectivity of a CFTR. A modulator may also change the ability of another modulator to affect the function of a CFTR. A modulator may act upon all or upon a specific subset of different forms (e.g., mutant forms) of CFTR. Modulators include, but are not limited to, potentiators, correctors, activators, inhibitors, agonists, antagonists, and blockers. Modulators also include protein trafficking correctors.

The phrase “functional CFTR” refers to a CFTR that responds to a known activator (such as apigenin, forskolin or IBMX—[3-isobutyl-1-methylxanthine]) or a known inhibitor (such as chromanol 293B, glibenclamide, lonidamine, NPPB—[5-nitro-2-(3-phenylpropylamino) benzoic acid], DPC—[diphenylamine-2-carboxylate] or niflumic acid) or other known modulators (such as 9-AC—[anthracene-9-carboxylic acid], or chlorotoxin) in substantially the same way as CFTR in a cell that normally expresses CFTR without engineering. CFTR behavior can be determined by, for example, physiological activities, and pharmacological responses. Physiological activities include, but are not limited to, chloride ion conductance. Pharmacological responses include, but are not limited to, activation by forskolin alone, or a mixture of forskolin, apigenin and IBMX [3-isobutyl-1-methylxanthine].

A “heterologous” or “introduced” CFTR protein means that the CFTR protein is encoded by a polynucleotide introduced into a host cell.

This invention relates to novel cells and cell lines that have been engineered to express CFTR. In some embodiments, the novel cells or cell lines of the invention express a functional, wild type CFTR (e.g., SEQ ID NO: 2). In some embodiments, the CFTR is a mutant CFTR (e.g., CFTR ΔF508; SEQ ID NO: 7). Illustrative CFTR mutants are set forth in Tables 1 and 2 (These tables are compiled based on mutation information obtained from a database developed by the Cystic Fibrosis Genetic Analysis Consortium available at www.genet.sickkids.on.ca/cftr/Home). According to the invention, the CFTR can be from any mammal, including rat, mouse, rabbit, goat, dog, cow, pig, or primate (e.g., human). In some embodiments, the novel cells or cell lines express an introduced functional CFTR (e.g., CFTR encoded by a transgene). In some embodiments, the novel cells or cell lines express a naturally-occurring CFTR, encoded by an endogenous CFTR gene that has been activated by gene activation technology. In preferred embodiments, the cells and cell lines stably express CFTR. The CFTR-expressing cells and cell lines of the invention have enhanced properties compared to cells and cell lines made by conventional methods. For example, the CFTR cells and cell lines have enhanced stability of expression (even when maintained in culture without selective pressure such as antibiotics) and possess high Z′ values in cell-based assays. The cells and cell lines of the invention provide detectable signal-to-noise signals, e.g., a signal-to-noise signal greater than 1:1. The cells and cell lines of the invention provide reliable readouts when used in high-throughput assays such as membrane potential assays, producing results that can match those from assays that are considered gold-standard in the field but too labor-intensive to become high-throughput (e.g., electrophysiology assays). In certain embodiments, the CFTR does not comprise a polypeptide tag.

TABLE 1
CFTR Mutants

		Location
		of
Name	Nucleotide Change	Mutation*	Consequence
1001+ 11C/T	C or T at 1001+ 11	intron 6b	sequence variation
1001+ 12C/T	C or T at 1001+ 12	intron 6b	sequence variation
1001+ 3A>T	A to T at 1001+ 3	intron 6b	Alternative splicing and
			complete skipping of exon 6b
1001+ 4A- >C+		intron 6b	splicing
993delCTTAA
1002- 2A>G	A to G at 1002- 2	6b	mRNA splicing defect
1002- 3T- >G	T to G at 1002- 3	intron 6b	mRNA splicing defect
1002- 56C/G	C or G at 1002- 56	intron 6b	sequence variation
1002- 7delTTT	Deletion of TTT beginning at	intron 6b	Interference with splicing
	1002- 7
1013delAA	deletion of AA from 1013	7	frameshift
-102T- >A	T to A at -102	promotor	regulatory mutation
1047C/T	C or T at 1047	7	sequence variation
1058delC	deletion of C at 1058	7	frameshift
1078delT	deletion of T at 1078	7	frameshift
107 G/A	G to A at 107	1	sequence variation
1086G/A	G or A at 1086	7	Sequence variation
1092A/G	A or G at 1092	7	sequence variation
1098G/A	G or A at 1098	7	sequence variation (Val at 322
			no change)
1104(C/G)	C or G at 1104	7	sequence variation
1112delT	deletion of T at 1112	7	frameshift
1119delA	deletion of A at 1119	7	frameshift
1138insG	insertion of G after 1138	7	frameshift
1150delA	deletion of A at 1150	7	frameshift
1150insTC	Insertion of TC at 1150	7	Frameshift
1151ins12	tandem duplication of	7	Insertion-duplication of 4
	12 bp from position		amino acids within the M6
	1140 to position 1151		domain (transmembrane
			domain)
1154insTC	insertion of TC after 1154	7	frameshift
1161delC	deletion of C at 1161	7	frameshift
1161insG	insertion of G after 1161	7	frameshift
1164 T/A	T to A at 1164	7	sequence variation
1185delTC	Deletion of TC at 1185	7	Frameshift
1199delG	deletion of G at 1199	7	frameshift
120del23	Deletion of 23 by from	promotor,	This mutation abolishes the
	nucleotide + 120 of	1	initiation codon at position
	exon 1 promoter, to		133. The next possible
	nucleotide 142 (the		initiation codon is located at
	first nucleotide of		intron 1 position 185 + 63.
	codon 4)
1213delT	deletion of T at 1213	7	frameshift
1215delG	deletion of G at 1215	7	frameshift
1221delCT	deletion of CT from 1221	7	frameshift
1233A/T	A or T at 1233	7	Sequence variation
1243ins6	insertion of ACAAAA after 1243	7	insertion of Asp and Lys after
			Lys370
1248+ 17C- >T	C or T at 1248+ 17	intron 7	sequence variation
1248+ 1G- >A	G to A at 1248+ 1	intron 7	mRNA splicing defect
1248+ 1G- >C	G to C at 1248+ 1	intron 7	Splicing
1248+ 31 A/C	1248 + 31 A>C	intron 7	sequence variation
1248+ 52T/C	T or C at 1248+ 52	intron 7	sequence variation
1249- 27delTA	deletion of TA at 1249- 27	intron 7	mRNA splicing defect
1249- 30delAT	deletion of AT from 1249- 30	intron 7	mRNA splicing defect
1249- 31A- >G	1249- 31 A>G	intron 7	mRNA splicing defect
1249- 5A- >G	A to G at 1249	intron 7	mRNA splicing defect
1249- 82C/T	C or T at 1249- 82	intron 7	sequence variation
124del23bp	delete 23 by from 124 to 146	1
1259insA	insertion of A after 1259	8	frameshift
125G/C	G or C at 125	1	sequence variation
1283delA	deletion of A at 1283	8	frameshift
1288insTA	Insertion of TA at 1285 Or	8	Frameshift
	Insertion of AT at 1284
1289insTA	Insertion of TA at 1289	8	Frameshift
1291delTT	delete TT from 1291	8	Frame shift
1294del7	deletion of 7 by from 1294	8	frameshift
1296G/T	G to T at 1296	8	sequence variation (Thr at 388
			no change)
129G/C	G or C at 129	1	sequence variation
1309delG	deletion of G at 1309	8	frameshift
1323insA	insertion of A after 1323	8	frameshift
1341+ 18A- >C	A to C at 1341+ 18	intron 8	mRNA splicing defect
1341+ 1G- >A	G to A at 1341+ 1	intron 8	mRNA splicing defect
1341+ 28C>T	C > T at 1341+ 28	intron 8	polymorphism
1341+ 28C/T	C or T at 1341+ 28	intron 8	sequence variation
1341+ 6 A- >G	A to G at 1341+ 6		mRNA splicing defect
1341+ 6 A- >G	A to G at 1341+ 6	intron 8	mRNA splicing defect
1341+ 79 C/T	1341 + 79 C- >T	intron 8	sequence variation
1341G- >A	G to A at 1341	8	sequence variation
1342- 11TTT- >G	TTT to G at 1342- 11	intron 8	mRNA splicing defect
1342- 12(GT)n	variable number of	intron 8	sequence variation
	copies (8- 10x)
	at around
	1342- 12 to - 35
1342- 13G/T	G or T at 1342- 13	intron 8	sequence variation
1342- 1delG	Deletion of G at 1342- 1	Intron 8	Frameshift
1342- 1G- >C	G to C at 1342- 1	intron 8	mRNA splicing defect
1342- 265(GT)n	variable number of copies at	intron 8	sequence variation (greater
	around 1342- 265 to - 310		than 8 alleles)
1342- 2A- >C	A to C at 1342- 2	intron 8	mRNA splicing defect
1342- 2delAG	deletion of AG from 1342- 2	intron 8	mRNA splicing defect
135del120ins300		1
1366delG	deletion of G at 1366	9	frameshift
1367del5	deletion of CAAAA at 1367	9	frameshift
1367delC	deletion of C at 1367	9	frameshift
1429del7bp	deletion of 17 bp from 1429	19	stop codon at amino acid 441
1460delAT	deletion of AT from 1460	9	frameshift
1461ins4	insertion of AGAT after 1461	9	frameshift
1461 T/C	T to C at 1461	9	sequence variation
1471delA	deletion of A at 1471	9	frameshift
1491- 1500del	Deletion between	9	Large in/del
	1491 to 1500
1497delGG	deletion of GG at 1497	9	frameshift
1504delG	deletion of G at 1504	9	frameshift
1524+ 1G- >A	G to A at 1524+ 1	intron 9	splice mutation
1524+ 60 insA	Ins A at 1524+ 60	intron 9	sequence variation
1524+ 68 G/A	1524 + 68 G>A	intron 9	sequence variation
1524+ 6insC	insertion of C after	intron 9	mRNA splicing defect
	1524+ 6, with
	G to A at 1524 + 12
1525- 18G/A	G or A at 1525- 18	intron 9	sequence variation or mRNA
			splicing defect
1525- 1G- >A	G to A at 1525- 1	intron 9	mRNA splicing defect
1525- 2A- >G	A to G at 1525- 2	intron 9	Splicing
1525- 47T- >G	1525- 47T>G	Intron 9	Sequence Variation
1525- 60G/A	G or A at 1525- 60	intron 9	sequence variation
1525- 61A/G	A or G at 1525- 61	intron 9	sequence variation
1531C/T (L467F)	C or T at 1531	10	sequence variation
1540del10	deletion of 10 bp after 1540	10	frameshift
1548delG	deletion of G from	10	frameshift
	1548 - 1550
1565 del CA	deletion of CA from 1565	10	frameshift
156G/A	G or A at 156	1	sequence variation
1571delG	deletion of G at 1571	10	frameshift
1572T/C	T or C at 1572	10	sequence variation
1576insT	insertion of T at 1576	10	framshift
1601delTC	deletion of TC from 1601	10	frameshift
	or CT from 1602
1609delCA	deletion of CA from 1609	10	frameshift
1612delTT	deletion of TT from 1612	10	frameshift
163G/A	G or A at 163	1	sequence variation
1650C/G	C to G at 1650	10	Ile to Met at 506;
			sequence variation
1651A/G	A or G at 1651	10	sequence variation
1653C/T	C to T at 1653	10	NO AMINOACID CHANGE
1660delG	Deletion of G at 1660	10	frameshift
1677delTA	deletion of TA from 1677	10	frameshift
1693A- >C	A to C at 1693	10	Ile to Leu at 521
			(sequence variation)
1706del17	deletion of 17 bp	10	deletion of splice site
	from 1706
1713A/G	A or G at 1713	10	sequence variation
1716+ 12T/C	T or C at 1716+ 12	intron 10	sequence variation
1716+ 13G/T	G or T at 1716+ 13	intron 10	sequence variation
1716+ 1G- >A	G to A at 1716+ 1	intron 10	mRNA splicing defect
1716+ 1G- >T	1716+ 1 G>T	intron 10	mRNA splicing defect
1716+ 2T- >C	T to Cat 1716+ 2	intron 10	mRNA splicing defect
1716+ 4 A- >T	1716+ 4 A>T	intron 10	mRNA splicing defect
1716+ 63ins11nt	insertion of 11	intron 10	sequence variation
	nucleotides after
	1716+ 63
1716+ 64A/C	A or C at 1716+ 64	intron 10	sequence variation
1716+ 77A/G	A or G at 1716+ 77	intron 10	sequence variation
1716+ 85C/T	C or T at 1716+ 85	intron 10	sequence variation
1716G/A	G or A at 1716	10	sequence variation
1717- 19T/C	T or C at 1717- 19	intron 10	sequence variation
1717- 1G- >A	G to A at 1717- 1	intron 10	mRNA splicing defect
1717- 2A- >G	A to G at 1717- 2	intron 10	mRNA splicing defect
1717- 3T- >G	T to G at 1717- 3	intron 10	mRNA splicing defect
1717- 8G- >A	G to A at 1717- 8	intron 10	mRNA splicing defect
1717- 9T- >A	T to A at 1717- 9	intron 10	mRNA splicing mutation
1742delAC	deletion of AC from 1742	11	frameshift
1749insTA	insertion of TA at 1749	11	frameshift resulting in
			premature termination at 540
174delA	deletion of A between	1	frameshift
	172- 174
175delC	deletion of C at 175	1	frameshift
175insT	insertion of T after 175	1	frameshift
1764T/G	T or G at 1764	11	sequence variation
1767del6	delete 6 nucleotide	11	In frame in/del
	from 1767
1773A/T	A or T at 1773	11	sequence variation
1774delCT	deletion of CT from 1774	11	frameshift
1782delA	deletion of A at 1782	11	frameshift
1784delG	deletion of G at 1784	11	frameshift
1787delA	deletion of A at position	11	frameshift, stop codon at 558
	1787 or 1788
1802delC	deletion of C at 1802	11	frameshift
1806delA	deletion of A at 1806	11	frameshift
1811+ 11A- >G	A to G at 1811+ 11	intron 11	Splicing
1811 + 1650 T>A	1811+ 1650 T>A	intron 11	Sequence variation
1811+ 1.6 kbA- >G	A to G at 1811+ 1.2 kb	intron 11	creation of splice donor site
1811+ 16T- >C	1811+ 16 T>C	intron 11	This mutation may lead to an
			alternative splicing, with the
			donor splice site located at
			nucleotide +18. This
			alternative splice site with
			the mutation at +16 has a
			higher PCU than the
			previously described
			mutation 1811 + 18G->A.
1811+ 18G- >A	G to A at 1811+ 18	intron 11	mRNA splicing defect
1811 + 1 G>A	G to A at 1811+ 1	intron 11	Splicing defect
1811+ 1G- >C	G to C at 1811+ 1	intron 11	mRNA splicing defect
1811+ 24G- >A	G to A at 1811 + 24	Intron 11	mRNA splicing defect
1811+ 34 G>A	G to A at 1811+ 34	intron 11	mRNA splicing defect
1811+ 5A- >G	1811 + 5 A>G	intron 11	mRNA splicing defect
1812- 108T/C	T or C at 1812- 108	intron 11	sequence variation
1812- 136T/C	T or C at 1812- 136	intron 11	sequence variation
1812- 1G- >A	G to A at 1812- 1	intron 11	mRNA splicing defect
1812- 26T- >C	T to C at 1812- 26	intron 11	splicing mutation
1812- 59T/G	T or G at 1812- 59	intron 11	sequence variation
1812- 5 T- >A	1812 - 5 T>A	intron 11	splicing mutation
1812- 99 T- >C	C to T at 1812- 99	Intron 11	Sequence Variation
1813insC	insertion of C after	12	frameshift
	1813 (or 1814)
182delT	deletion of T at 182	1	frameshift
1833delT	deletion of T at 1833	12	frameshift
1845delAG/1846delGA	deletion of AG at 1845	12	frameshift
	or GA at 1846
185+ 1G- >T	G to T at 185+ 1	intron 1	mRNA splicing defect
185+ 45A- >G	A to G at 185+ 45	intron 1	sequence variation
185+ 4A- >T	A to T at 185+ 4	intron 1	mRNA splicing defect
			(CBAVD)
186- 13C- >G	C to G at 186- 13	intron 1	mRNA splicing defect
1870delG	deletion of G at 1870	12	frameshift
1874insT	insertion of T between	12	frameshift
	1871 and 1874
1898+ 152T/A	T or A at 1898+ 152	intron 12	sequence variation
1898+ 1G- >A	G to A at 1898+ 1	intron 12	mRNA splicing defect
1898+ 1G- >C	G to C at 1898+ 1	intron 12	mRNA splicing defect
1898+ 1G- >T	G to T at 1898+ 1	intron 12	mRNA splicing defect
1898+ 30G/A	G or A at 1898+ 30	intron 12	sequence variation
1898+ 3A- >C	A to C at 1898+ 3	intron 12	mRNA splicing defect
1898+ 3A- >G	A to G at 1898+ 3	intron 12	mRNA splicing defect
1898+ 5G- >A	G to A at 1898+ 5	intron 12	mRNA splicing defect
1898+ 5G- >T	G to T at 1898+ 5	intron 12	mRNA splicing defect
1898+ 73T- >G	T to G at 1898+ 73	intron 12	mRNA splicing defect
1918delGC	deletion of GC from 1918	13	frameshift
1924del7	deletion of 7 by (AAACTA)	13	frameshift
	from 1924
1932delG	Deletion of G at	13	Frameshift a premature stop
	nucleotide 1932		codon appears 10 codons
			further.
1949del84	deletion of 84 bp	13	deletion of 28 a.a.
	from 1949		(Met607 to Gln634)
2003del8	Deletion of GCTATTTT	13	Frameshift
	from 2003
2043delG	deletion of G at 2043	13	frameshift
2051delTT	deletion of TT from 2051	13	frameshift
2055del9- >A	deletion of 9 bp	13	frameshift
	CTCAAAACT to A
	at 2055
2064C/G	C or G at 2064	13	sequence variation (Leu at
			644 no change)
2082C/T	C or T at 2082	13	sequence variation (no
			change Phe at 650)
2092A/G	A or G at 2092	13	sequence variation
2104insA+ 2109-	insertion of A at	13
2118del10	2104, deletion of
	10 bp at 2109
2105-	Deletion of 13 bp and	13	Frameshift
2117de113insAGAAA	insertion of
	AGAAA at 2105- 2117
2108delA	deletion of A at 2108	13	frameshift
2113delA	deletion of A at 2113	13	frameshift
2116delCTAA	deletion of CTAA at 2116	13	frameshift
2118del4	deletion of AACT from 2118	13	frameshift
211delG	deletion of G at 211	2	frameshift
2141insA	insertion of A after 2141	13	frameshift
2143delT	deletion of T at 2143	13	frameshift
2176insC	insertion of C after 2176	13	frameshift
2183AA- >G	A to G at 2183 and deletion	13	frameshift
	of A at 2184
2183delAA	deletion of AA at 2183	13	frameshift
2184A/G	A to G at 2184	13	no change
2184delA	deletion of A at 2184	13	frameshift
2184insA	insertion of A after 2184	13	frameshift
2185insC	insertion of C at 2185	13	frameshift
2193ins4	Insertion of 4T at 2193	13	Frameshift
2215insG	insertion of G at 2215	13	frameshift
2221insA	insertion of A at 2221	13	Frameshift a premature stop
			codon appears 33 codons
			further
2238C/G	C or G at 2238	13	sequence variation
223C/T	C or T at 223	2	sequence variation
2289- 2295del7bpinsGT	Deletion of 7 bp	13	Frameshift
	and insertion of
	GT at 2289- 2295
2307insA	insertion of A after 2307	13	frameshift
232del18	Deletion of 18 bp from 232	2	Deletion of 6 aa from
			Leu34 to Gln39
2335delA	deletion of A at 2335	13	frameshift
2347delG	deletion of G at 2347	13	frameshift
2372del8	deletion of 8 by from 2372	13	frameshift
2377C/T	C or T at 2377	13	sequence variation (no
			change for Leu at 749)
237insA	insertion of A after 237	2	frameshift
2380_2387del	Deletion of 8 bp from 2380	13	Frameshift
2391 C/T	2391 C>T	13	Polymorphism
2406delCC	deletion of CC at 2406	13	Frameshift
2409delC	Deletion of C at 2409	13	Frameshift
2412G/A	G to A at 2412	13	Sequence variation
2418GG>T	G to T at 2418	13	missense
241delAT	deletion of AT from 241	2	frameshift
2423delG	deletion of G at 2423	13	frameshift
244delTA	deletion of TA from 244	2	frameshift
2456delAC	deletion of AC at 2456	13	frameshift
2493ins8	insertion of 8bp after 2493	13	frameshift
2512delG	Deletion of G at 2512	13	Frameshift
2522insC	insertion of C after 2522	13	frameshift
2553A/G	A or G at 2553	13	sequence variation
2556insAT	insertion of AT after 2556	13	frameshift
2566insT	insertion of T after 2566	13	frameshift
2585delT	deletion of T at 2585	13	stop codon at amino acid 820
2603delT	deletion of T at 2603/4	13	frameshift
2622+ 14G/A	G or A at 2622+ 14	intron 13	sequence variation
2622+ 1G- >A	G to A at 2622+ 1	intron 13	mRNA splicing defect
2622+ 1G- >T	G to T at 2622+ 1	intron 13	splice mutation
2622+ 2del6	deletion of TAGGTA	intron 13	mRNA splicing defect
	from 2622+ 2
2622+ 2T>C	T to C at 2622+ 2	intron 13	mRNA splicing defect
2623- 11 C- >T	2623- 11 C>T	intron 13	Polymorphism
2623- 23A- >G	2623- 23 A>G	intron 13	mRNA splicing defect
2623- 2A- >G	A to G at 2623- 2	intron 23	Splicing
2634delT	Deletion of T at 2634	14a	frameshift
2634insT	insertion of T after 2634	14a	frameshift
263A/T	A or T at 263	2	sequence variation
2640delT	deletion of T at 2640	14a	frameshift
2691T/C	T or C at 2691	14a	sequence variation
2694delT	deletion of T at 2694	14a	frameshift
2694T/C	T or C at 2694	14a	sequence variation
2694T/G	T or G at 2694	14a	sequence variation
2703G/A	G or A at 2703	14a	sequence variation (Lys
			at 857 no change)
2711delT	deletion of T at 2711	14a	frameshift
2721del11	deletion of 11 bp from 2721	14a	frameshift
2723delTT	deletion of TT from 2723	14a	frameshift
2732insA	insertion of A at 2732	14a	frameshift
2734G- >AT	Deletion of G at 2734	14a	frameshift
	with insertion of AT
2736G/A	G or A at 2736	14a	sequence variation
2747delC	Deletion of C at	14a	Frameshift a premature stop
	nucleotide 2747		codon appears 34 codons
			further
2751+ 2T- >A	T to A at 2751+ 2	intron	mRNA splicing defect
		14a
2751+ 3A- >G	A to G at 2751+ 3	intron	mRNA splicing defect
		14a	(CBAVD)
2751G- >A	G to A at 2751	14a	mRNA splicing defect
2752- 15C/G	C or G at 2752- 15	intron	sequence variation
		14a
2752- 17G/A	G to A at 2752- 17	intron	sequence variation
		14a
2752- 1G- >C	G to C at 2752- 1	intron	splice mutation
		14a
2752- 1G- >T	G to T at 2752- 1	intron	mRNA splicing defect
		14a
2752- 22A/G	A or G at 2752- 22	intron	sequence variation
		14a
2752- 26A- >G	A to G at 2752- 26	intron	mRNA splicing defect
		14a
2752- 2A>G	A to G at 2752- 2	Intron	mRNA splicing defect
		14a
2752- 674_3499+	2752- 674_3499+	14b, 15,	Large deletion removing
198del9855	198del9855bp	16, 17a,	exons 14b to 17b.
		17b	Frameshift
2752- 6T- >C	T to C at 2752- 6	intron	Splicing
		14a
2752- 97C- >T	C to T at 2752- 97	intron	Splicing
		14a
2766del8	deletion of 8 bp from 2766	14b	frameshift
2787del16	Deletion of 16	14b,	Splicing mutation.
	nucleotides from	intron
	2787	14b
2789+ 2insA	insertion of A	intron	mRNA splicing defect (CAVD)
	after 2789+ 2	14b
2789+ 32T/C	T or C at 2789+ 32	intron	sequence variation
		14b
2789+ 3delG	deletion of G at 2789+ 3	intron	mRNA splicing defect
		14b
2789+ 5G- >A	G to A at 2789+ 5	intron	mRNA splicing defect
		14b
2790- 108G/C	G or C at 2790- 108	intron	sequence variation
		14b
2790- 1G- >C	G to C at 2790- 1	intron	mRNA splicing defect
		14b
2790- 1G- >T	G to T at 2790- 1	intron	mRNA splicing defect
		14b
2790- 21G/A	G or A at 2790- 21	intron	sequence variation
		14b
2790- 2A- >G	A to G at 2790- 2	intron	mRNA splicing defect
		14b
279A/G	A to G at 279	2	No change (Leu at 49)
2811G/T	G or T at 2811	15	sequence variation
2819del4bpins13bp	delete 4bp(CTCA) at	15	Thr to Met at 896, His to Ser
	2819, insert 13 bp		at 897, insertion of Thr, Met
	(TGAGTACTATGAG (SEQ		and Ser after 897
	ID NO: 10)) at 2819
2839T/C	T or C at 2839	15	sequence variation
2844A/T	A or T at 2844	15	sequence variation (Ala at 904
			no change)
284delA	deletion of A at 284	2	frameshift
2851A/G	A or G at 2851	15	Ile or Val at 907
2856C/T	C or T at 2856	15	sequence variation (Thr at 908
			no change)
2858G/T	G or T at 2858	15	sequence variation
2868 G/A	G to A at 2868	15	sequence variation
2869insG	insertion of G after 2869	15	frameshift
2896insAG	insertion of AG after 2896	15	frameshift
2901C/T	C or T at 2901	15	sequence variation
2907delTT	deletion of TT from 2907	15	frameshift
2909delT	deletion of T at 2909	15	frameshift
2940A/G	A or G at 2940	15	sequence variation
2942insT	insertion of T at 2942	15	frameshift resulting in
			premature termination at
			codon 974
2948AT- >C	AT to C at 2948	15	frameshift resulting in
			premature termination at 2953
295ins8	insertion of ATTGGAAA	2	frameshift
	after 295
296+ 128G/C	G or C at 296+ 128	intron 2	sequence variation
296+ 12T- >C	T to C at 296+ 12	intron 2	mRNA splicing defect
296+ 1G- >A	G to A at 296+ 1	intron 2	splicing
296+ 1G- >C	G to C at 296+ 1	intron 2	mRNA splicing defect
296+ 1G- >T	G to T at 296+ 1	intron 2	missense; mRNA splicing
			defect
296+ 28A- >G	A to G at 296+ 28	intron 2	mRNA splicing
296+ 2T- >A	T to A at 296+ 2	intron 2	mRNA splicing Defect
296+ 2T- >C	T to C at 296+ 2	intron 2	mRNA splicing defect
296+ 2T- >G	T to G at 296 + 2	intron 2	mRNA splicing defect
296+ 3insT	insertion of T	intron 2	mRNA splicing defect
	after 296+ 3
2967G/A	G or A at 2967	15	sequence variation (no
			change for Ser at 945)
296+ 9A- >T	A to T at 296+ 9	intron 2	mRNA splicing defect
297- 10T- >G	T to G at 297- 10	intron 2	splice mutation
297- 12insA	insertion of A at 297- 12	intron 2	splice mutation
297- 28insA	insertion of A	intron 2	mRNA splicing defect
	after 297- 28
297- 2A- >G	A to G at 297- 2	intron 2	mRNA splicing defect
297- 3C- >A	C to A at 297- 3	intron 2	mRNA splicing defect
297- 3C- >T	C to T at 297- 3	intron 2	mRNA splicing defect
297- 45 A- >G	A to G at 297- 45		Sequence variation
297- 50A/G	A or G at 297- 50	intron 2	sequence variation
297- 55C/T	C to T at 297- 55	intron 2	sequence variation
297- 57 G/T	297 - 57 G>T	intron 2	sequence variation
297- 67A/C	A or C at 297- 67	intron 2	sequence variation
297- 73 A/G	297 - 73 A>G	intron 2	sequence variation
2991del32	deletion of 32 bp from	15	frameshift
	2991 to 3022
3007delG	deletion of G at 3007	15	frameshift
300delA	deletion of A at 300	3	frameshift
3028delA	deletion of A at 3028	15	frameshift
3030G/A	G or A at 3030	15	sequence variation
3040+ 11A/T	3040+ 11 A>T	intron 15	Polymorphism
3040+ 23T- >C	T to C at 3040+ 23	intron 15	Splicing
3040+ 2T- >C	T to C at 3040+ 2	intron 15	mRNA splicing defect
3041- 11del7	deletion of GTATATT	intron 15	mRNA splicing mutation
	at 3041- 11
3041- 15T- >G	T to G at 3041- 15	intron 15	mRNA splicing mutation
3041- 1G- >A	G to A at 3041- 1	intron 15	mRNA splicing defect
3041- 4A- >G	A to G at 3041- 4	intron 6b	splicing
3041- 51 T/G	3041 - 51 T>G	intron 15	sequence variation
3041- 52C/G	C or G at 3041- 52	intron 15	sequence variation
3041- 71G/C	G or C at 3041- 71	intron 15	sequence variation
3041- 92G/A	G or A at 3041- 92	intron 15	sequence variation
3041delG	deletion of G at 3041	16	frameshift
3056delGA	deletion of GA from 3056	16	frameshift
306delTAGA	deletion of TAGA from 306	3	frameshift
306insA	insertion of A at 306	3	frameshift
3079delTT	deletion of TT from 3079	16	frameshift
3100insA	insertion of A after 3100	16	frameshift
3120+ 198G- >A	G to A at 3120+ 198	intron 16	Splicing
3120+ 1G- >A	G to A at 3120+ 1	intron 16	mRNA splicing defect
3120+ 35 A- >T	A to T at 3120+ 35	Intron 16	mRNA splicing defect
3120+ 41delA	Delete A at 3120+ 41	intron 16	sequence variation
3120+ 45A/G	A or G at 3120+ 45	intron 16	sequence variation
3120G- >A	G to A at 3120	16	mRNA splicing defect
3121- 14C/A	C or A at 3121- 14	intron 16	Sequence variation
3121- 1G- >A	G to A at 3121- 1	intron 16	mRNA splicing defect
3121- 2A- >G	A to G at 3121- 2	intron 16	mRNA splicing defect
3121- 2A- >T	A to T at 3121- 2	intron 16	mRNA splicing defect
3121- 3C- >G	C to G at 3121- 3	intron 16	mRNA splicing
3121- 92A12/13	12A or 13A at 3121- 92	intron 16	sequence variation
3121- 977_3499+	3121- 977_3499+ 248del2515bp	17a, 17b	Large deletion removing
248del2515			exons 17a and 17b.
			Frameshift
3126del4	deletion of ATTA from 3126	17a	frameshift
3129del4	deletion of 4 bp from 3129	17a	frameshift
3130del15	delete 15 nucleotide at 3130	17a	In fram in/del
3130delA	Deletion of A at 3130	17a	frameshift
3131del15	deletion of 15 bp from	17a	deletion of Val at 1001
	3130, 3131, or 3132		to Ile at 1005
3132delTG	deletion of TG from 3132	17a	frameshift
3141del9	del AGCTATAGC from 3141	17a	Frameshift
3152delT	delete T at 3152	17a	frameshift
3153delT	deletion of T at 3153	17a	frameshift
3154delG	deletion of G at 3154	17a	frameshift
3171delC	deletion of C at 3171	17a	frameshift resulting in
			premature termination at 1022
3171insC	insertion of C after 3171	17a	frameshift
3173delAC	deletion of AC from 3173	17a	frameshift
3195del6	deletion of AGTGAT from	17a	deletion of Val 1022 and
	3195 to 3200		Ile 1023
3196del54	deletion of 54 bp	17a	deletion of 18 aa from codon
	from 3196		1022
3199del6	deletion of ATAGTG	17a	deletion of Ile at 1023
	from 3199		and Val at 1024
3200_3204delTAGTG	Deletion of TAGTG	17a	Frameshift
	from 3200
3238delA	3238delA	17a	frameshift
3271+ 101C/G	C or G at 3271+ 101	intron	sequence variation
		17a
3271+ 183 T to G	T to G at 3271+ 183	intron	sequence variation
		17a
3271+ 18C/T	C or T at 3271+ 18	intron	sequence variation
		17a
3271 +1G- >A	G to A at 3271+ 1	intron	mRNA splicing defect
		17a
3271+ 1G>T	G to T at 3271+ 1	Intron	mRNA splicing defect
		17a
3271+ 42A/T	A or T at 3271+ 42	intron	sequence variation
		17a
3271+ 80A/T	A or T at 3271+ 80	Intron	Sequence variation
		17a
3271+ 8A>G	A to G at 3271+ 8	intron	RNA splicing defect
		17a
3271delGG	deletion of GG at 3271	17a	framshift for exon 17b,
			loss of splice site
3272- 11A- >G	A to G at 3272- 11	intron	Splicing
		17a
3272- 1G- >A	G to A at 3272- 1	intron	mRNA splicing defect
		17a
3272- 26A- >G	A to G at 3272- 26	intron	mRNA splicing defect
		17a
3272- 33A/G	A or G at 3272- 33	intron	sequence variation
		17a
3272- 42 G/T	3272 - 42 G>T	intron	sequence variation
		17a
3272- 4A- >G	A to G at 3272- 4	intron	mRNA splicing defect
		17a
3272- 54del704	deletion of 704 bp	intron	deletion of exon 17b
	from 3272- 54	17a
3272- 93T/C	T or C at 3272- 93	intron	sequence variation
		17a
3272- 9A- >T	A to T at 3272- 9	intron	mRNA splicing defect
		17a
3293delA	deletion of A at 3293	17b	frameshift
- 329A/G	A or G at - 329 upstream	promotor	sequence variation
	of the cap site
3320ins5	insertion of CTATG	17b	frameshift
	after 3320
3333C/T	C or T at 3333	17b	sequence variation
3336C/T	C or T at 3336	17b	sequence variation
3359delCT	deletion of CT from 3359	17b	frameshift
3384A/G	A or G at 3384	17b	sequence variation
3396delC	deletion of C at 3396	17	frameshift
- 33G- >A	G to A at - 33	promotor	promoter mutation
3413del355_insTGTTAA	Partial deletion of	17b	A stop codon appears very
	exon 17b. It removes		early in the new sequence but
	355 bp, i.e. from nt		the consequences at the RNA
	3413 (in codon 1094)		level remain to be studied.
	to 3499+ 268 in
	intron 17b; the
	sequence “TGTTAA”
	is inserted at the
	breakpoints.
3417A/T	A or T at 3417	17b	sequence variation
3419delT	deletion of T at 3419	17b	frameshift
3423delC	deletion of C at 3423	17b	frameshift
3425delG	deletion of G at 3425 or 3426	17b	frameshift
3438A/G	A or G at 3438	17b	Sequence variation
3447delG	Deleletion of G at 3447	17b	Frameshift
345T/C	T or C at 345	3	sequence variation
3471T/C	T or C at 3471	17b	sequence variation
3477C/A	C or A at 3477	17b	sequence variation
347delC	deletion of C at 347	3	frameshift
3495delA	deletion of A at 3495	17b	frameshift
3499+ 29G/A	G or A at 3499+ 29	Intron	Sequence variation
		17b
3499+ 2T- >C	T to C at 3499+ 2	intron	mRNA splicing defect
		17b
3499+ 37G/A	G or A at 3499+ 37	intron	sequence variation
		17b
3499+ 3A- >G	A to G at 3499+ 3	intron	mRNA splicing defect
		17b
3499+ 45T/C	T or C at 3499+ 45	intron	sequence variation
		17b
3499+ 6A- >G	A to G at 3499+ 6	intron	mRNA splicing defect
		17b
3499+ 7T- >G	T to G at 3499+ 7	intron	Splicing
		17b
3500- 140A/C	A or C at 3500- 140	intron	sequence variation
		17b
3500 - 1 G to A	3500 - 1 G>A	intron	mRNA splicing defect
		17b
3500- 2A- >G	A to G at 3500- 2	intron	mRNA splicing defect
		17b
3500- 44G/A	G or A at 3500- 44	intron	sequence variation
		17b
3500- 50 A/C	3500 - 50 A>C	intron	sequence variation
		17b
3523A- >G	A to G at 3523	18	Ile to Val at 1131
3532AC- >GTA	AC to GTA from 3532	18	frameshift
3556insAGTA	insertion of AGTA after	18	frame shift
	position 3556
3577delT	deletion of T at 3577	18	frameshift
359insT	insertion of T after 359	3	frameshift
3600+ 2insT	insertion of T after 3600+ 2	intron 18	mRNA splicing defect
3600+ 2T- >C	T to C at 3600+ 2	intron 18	sequence variation
3600+ 42G/A	G or A at 3600+ 42	intron 18	sequence variation
3600+ 5G- >A	G to A at 3600+ 5	intron 18	mRNA splicing defect
3600G- >A	G to A at 3600	18	mRNA splicing defect
3601- 111G/C	G or C at 3601- 111	intron 18	sequence variation
3601- 17T- >C	T to C at 3601- 17	intron 18	mRNA splicing defect
3601- 20T- >C	T to C at 3601- 20	intron 18	mRNA splicing mutant
3601- 2A- >G	A to G at 3601- 2	intron 18	mRNA splicing defect
3601- 65C/A	C or A at 3601- 65	intron 18	sequence variation
360- 365insT	Insertion of T at 360- 365	3	Frameshift
360delT	deletion of T at 360	3	frameshift
3617delGA	Deletion of GA from 3617	19	Frameshift
3617G/T	G or T at 3617	19	sequence variation
3622insT	insertion of T after 3622	19	frameshift
3629delT	Deletion of T at 3629	19	Frame shift
3636 C/T	C to T at 3636	19	sequence variation (Asp at
			1168 no change)
- 363C/T	C to T at - 363	promotor	promoter mutation
365- 366insT (W79fs)	insertion at 360 - 365	3	Frameshift (W79fs)
3659delC	deletion of C at 3659	19	frameshift
3662delA	deletion of A at 3662	19	frameshift
3667del4	deletion of 4 bp from 3667	19	frameshift
3667ins4	insertion of TCAA after 3667	19	frameshift
3670delA	deletion of A at 3670	19	frameshift
3696G/A	G to A at 3696	18	No change to Ser at 1188
3724delG	deletion of G at 3724	19	frameshift
3726G/T	G or T at 3726	19	sequence variation
3732delA	deletion of A at 3732 and	19	frameshift and Lys to Glu at
	A to G at 3730		1200
3737delA	deletion of A at 3737	19	frameshift
3750delAG	deletion of AG from 3750	19	frameshift
3755delG	deletion of G between	19	frameshift
	3751 and 3755
3780 A/C	A to C at 3780	19	sequence variation
3789insA	insertion of A at 3789	19	frameshift resulting in a
			premature termination at 3921
3791C/T	C or T at 3791	19	sequence variation
3791delC	deletion of C at 3791	19	frameshift
379- 381insT	Insertion of T at 379- 381	3	Frameshift
3821- 3823del T	deletion of T at 3821- 3823	19	frameshift (Stop at 1234)
3821delT	deletion of T at 3821	19	frameshift
3849+ 10kbC- >T	C to T in a 6.2 kb	intron 19	creation of splice
	EcoRI fragment		acceptor site
	10 kb from 19
3849+ 1G- >A	G to A at 3849+ 1	intron 19	mRNA splicing defect
3849+ 40A- >G	A to G at 3849+ 40	intron 19	Splicing
3849+ 45G- >A	G to A at 3849+ 45	intron 19	Splicing
3849+ 4A- >G	A to G at 3849+ 4	intron 19	mRNA splicing defect
3849+ 5G- >A	G to A at 3849+ 5	intron 19	mRNA splicing defect
3849G- >A	G to A at 3849	19	mRNA splicing defect
3850- 129T/C	T or C at 3850- 129	intron 19	sequence variation
3850- 1G- >A	G to A at 3850- 1	intron 19	mRNA splicing defect
3850- 3T- >G	T to G at 3850- 3	intron 19	mRNA splicing defect
3850- 41C/G	3850- 41 C>G	intron 19	Sequence variation
3850- 79T/C	T or C at 3850- 79	intron 19	sequence variation
3860ins31	insertion of 31 bp	20	frameshift
	after 3860
3867A/G	A or G at 3867	20	sequence variation
3876delA	deletion of A at 3876	20	frameshift
3878delG	deletion of G at 3878	20	frameshift mutation at 1249
			and stop codon at 1258
3891 G/A	G or A at 3891	20	Sequence Variation
3898insC	insertion of C after 3898	20	frameshift
3905insT	insertion of T after 3905	20	frameshift
3906insG	insertion of G after 3906	20	frameshift
3922del10- >C	deletion of 10 bp from	20	deletion of Glu 1264 to
	3922 and replacement		Glu 1266
	with 3921
3939C/T	C or T at 3939	20	sequence variation
3944delGT	deletion of GT from 3944	20	frameshift
394delTT	deletion of TT from 394	3	frameshift
3960- 3961delA	Deletion of A at	20	Frameshift
	3960- 3961
4005+ 117T/G	T or G at 4005+ 117	intron 20	sequence variation
4005+ 121delTT	8T or 6T at 4005+ 121	intron 20	sequence variation
4005+ 1G- >A	G to A at 4005+ 1	intron 20	mRNA splicing defect
4005+ 23delA	Deletion of A	Intron 20	Sequence variation- mRNA
	at 4005+ 23		splicing defect
4005+ 28insA	6A or 7A at 4005+ 28	intron 20	sequence variation
4005+ 29G- >C	G to C at 4005+ 29	intron 20	Splicing
4005+ 2T- >C	T to C at 4005+ 2	intron 20	mRNA splicing defect
4005+ 33A- >G	A to G at 4005+ 33	intron 20	Splicing
4006- 103delT	deletion of T	intron 20	sequence variation
	at 4006- 103
4006- 11 t- >G	T to G at 4006- 11		mRNA splicing defect
4006- 14C- >G	C to G at 4006- 14	intron 20	mRNA splicing defect
4006- 19del3	deletion of 3 bp	intron 20	mRNA splicing defect
	from 4006- 19
4006- 200G/A	G or A at 4006- 200	intron 20	sequence variation
4006- 26 T/C	4006 - 26 T>C	intron 20	sequence variation
4006- 46delTATTT	Deletion from 4006- 46	intron 20	Splicing defect
	to 4006- 42
4006- 4A- >G	A to G at 4006- 4	intron 20	mRNA splicing defect
4006- 50 A/C	4006 - 50 A>C	intron 20	sequence variation
4006- 61del14	deletion of 14 bp from	intron 20	mRNA splicing defect
	4006- 61 to 4006- 47
4006- 8T- >A	T to A at 4006- 8	intron 20	mRNA splicing defect
4006delA	deletion of A at 4006	21	frameshift
4010del4	deletion of TATT	21	frameshift
	from 4010
4015delA	deletion of A at 4015	21	frameshift
4016insT	insertion of T at 4016	21	frameshift
4022insT	insertion of T at 4022	21	Frameshift.
4029A/G	A or G at 4029	21	sequence variation
4040delA	deletion of A at 4040	21	frameshift
4041_4046del6insTGT	Deletion of nucleotides	21	deletion of Leu at 1304
	4041 to 4046 and		and Asp at 1305,
	insertion of TGT		insertion of Val at 1304
4048insCC	insertion of CC	21	frameshift
	after 4048
405+ 1G- >A	G to A at 405+ 1	intron 3	mRNA splicing defect
405+ 3A- >C	A to C at 405+ 3	intron 3	mRNA splicing defect
405+ 42A/G	A or G at 405+ 42	intron 3	sequence variation
405+ 46G/T	G or T at 405+ 46	intron 3	sequence variation
405+ 4A- >G	A to G at 405+ 4	intron 3	mRNA splicing defect
406- 10C- >G	C to G at 406- 10	intron 3	mRNA splicing defect
406- 112T/A	T or A at 406- 112	intron 3	sequence variation
406- 13T/C	T or C at 406- 13	intron 3	sequence variation
406- 1G- >A	G to A at 406- 1	intron 3	mRNA splicing defect
406- 1G- >C	G to C at 406- 1	intron 3	mRNA splicing defect
406- 1G- >T	G to T at 406- 1	intron 3	mRNA splicing defect
406- 2A- >C	A to C at 406- 2	intron 3	mRNA splicing defect
406- 2A- >G	A to G at 406- 2	intron 3	mRNA splicing defect
406- 3T- >C	T to C at 406- 3	intron 3	mRNA splicing defect
406- 5T- >G	T to G at 406- 5	intron 3	mRNA splicing defect
406- 6T- >C	T to C at 406- 6	intron 3	mRNA splicing defect
406- 82T/A	T or A at 406- 82	Intron 3	Sequence variation
406- 83A/G	A or G at 406- 83	intron 3	sequence variation
4086T/C	T or C at 4086	21	sequence variation
4095+ 1G>C	4095+ 1 G>C	intron 21	mRNA splicing defect
4095+ 1G- >T	4095+ 1 G>T	Intron 21	mRNA splicing defect
4095+ 2T- >A	4095+ 2 T>A	intron 21	mRNA slicing defect
4095+ 42T/C	T or C at 4095+ 42	intron 21	sequence variation
4096- 1G- >A	G to A at 4096- 1	intron 21	mRNA splicing defect
4096- 283T/C	T or C at 4096- 283	intron 21	sequence variation
4096- 28G- >A	G to A at 4096- 28	intron 21	mRNA splicing defect
4096- 3C- >G	C to G at 4096- 3	intron 21	mRNA splicing defect
40G/C	G to C at 40	1	Sequence variation
4108delT	deletion of T at 4108	22	frameshift
4114ATA- >TT	ATA to TT from 4114	22	Ile to Leu at 1328 and
			frameshift
412del7- >TA	deletion of ACCAAAG	4	frameshift
	from 412 and
	insertion of TA
4168delCTAAGCC	Deletion of CTAAGCC	22
	at 4168
4171insA	insertion of A at 4171	22	Frameshift a premature stop
			codon appears 12 codons
			further.
4172delGC	deletion of GC from 4172	22	frameshift
4173delC	deletion of C at 4173	22	frameshift
4203TAG- >AA	TAG to AA at 4203	22	frameshift
4209TGTT- >AA	TGTT to AA from 4209	22	Frame shift
4218insT	insertion of T after 4218	22	frameshift
4269- 108A- >G	A to G at 4269- 108	intron 22	sequence variation
4269- 139G/A	G or A at 4269- 139	intron 22	sequence variation
4271delC	deletion of C at 4271	23	frameshift
4272delA	Deletion of nucleotide	23	Frameshift
	A at 4272 position
4279insA	insertion of A after 4279	23	frameshift
4301)delA	deletion of A at 4301	23	frameshift
	or 4302
4326delTC	Deletion of TC from	23	FrameShift
	4326 to 4327
4326delTC	deletion of TC from 4326	23	frameshift
4329C/G	C or G at 4329	Exon 23	Sequence Variation
4332delTG	deletion of TG at 4332	23	framshift
4356G/A	G or A at 4356	23	sequence variation
435insA	insertion of A after 435	4	frameshift
4374+ 10T- >C	T to C at 4374+ 10	intron 23	splicing
4374+ 13A/G	A or G at 4374+ 13	intron 23	sequence variation
4374+ 14A/G	A or G at 4374+ 14	intron 23	sequence variation
4374+ 1G- >A	G to A at 4374+ 1	intron 23	mRNA splicing defect
4374+ 1G- >T	G to T at 4374+ 1	intron 23	mRNA splicing defect
4374_4374+ 1GG>TT	4374_4374+ 1GG>TT	23,	mRNA splicing defect
		intron 23
4375- 15C/T	C or T at 4375- 15	intron 23	sequence variation
4375- 1G- >C	G to C at 4375- 1	intron 23	splicing mutation
4375- 36delT	deletion of T at 375- 36	intron 23	sequence variation
4382delA	deletion of A at 4382	24	frameshift
4404C/T	C or T at 4404	24	sequence variation
441delA	deletion of A at 441	4	frameshift
	and T to A at 486
4428insGA	insertion of GA	24	frameshift
	after 4428
444delA	deletion of A at 444	4	frameshift
4464 C/T	C to T at 4464	24	sequence variation
451del8	deletion of GCTTCCTA	4	frameshift
	from 451
4521G/A	G or A at 4521	24	sequence variation
4557 G/A	G to A at 4557	24	sequence variation (Leu at
			1475 no change)
4563T/C	T or C at 4563	24	sequence variation
4575+ 2G- >A	G to A at 4575+ 2	intron 24	Splicing
457TAT- >G	TAT to G at 457	4	frameshift
458delAT	deletion of AT at 458	4	frameshift
4608- 4638del31	31 bp deletion between	intron 24	sequence variation
	4608 and 4638
460delG	deletion of G at 460	4	frameshift
- 461A- >G	A to G at - 461	promotor	Sequence variation
4655T- >G	T to G at 4655	intron 24	sequence variation
465G/A	G or A at 465	4	sequence variation
4700T8/9	8T or 9T at 4700	intron 24	sequence variation
- 471delAGG	deletion of AGG	promotor	promoter mutation
	from - 471
489 C/T	C to T at 489	4	sequence variation
489delC	deletion of C at 489	4	frameshift
48C/G	C or G at 48	promotor	sequence variation
492G/A	G or A at 492	4	sequence variation
519delT	T deleted	4	frameshift
525delT	deletion of T at 525	4	frameshift
541del4	deletion of	4	frameshift
	CTCC from 541
541delC	deletion of C at 541	4	frameshift
545T/C	T or C at 545	4	sequence variation
546insCTA	insertion of	4	frameshift
	CTA at 546
547insGA	insertion of GA	4	Frameshift; a premature
	between nucleotides		stop codon appears 15
	547 and 548		codons further.
547insTA	insertion of TA	4	frameshift
	after 547
549C/T	C to T at 549	4	sequence variation
			(His at 139 no change)
552insA	insertion of A	4	frameshift
	after 552
556delA	deletion of A at 556	4	frameshift
557delT	deletion of T at 557	4	frameshift
565delC	deletion of C at 565	4	frameshift
574delA	deletion of A at 574	4	frameshift
576InsCTA	Insert CTA at 576	4	In frame in/del
- 589G/A	G or A at - 589	Promoter	Sequence Variation
591del18	deletion of 18 bp	4	deletion of 6 a.a. from
	from 591
605insT	insertion of T	4	frameshift
	after 605
612T/A	T or A at 612	4	sequence variation
	(together
	with Y161S)
621+ 1G- >T	G to T at 621 +1	intron 4	mRNA splicing defect
621+ 2T- >C	T to C at 621 +2	intron 4	mRNA splicing defect
621+ 2T- >G	T to G at 621 +2	intron 4	mRNA splicing defect
621+ 31C/G	C or G at 621 +31	intron 4	sequence variation
621+ 3A- >G	A to G at 621 +3	intron 4	mRNA splicing defect
621G- >A	G to A at 621	4	mRNA splicing defect
622- 103A/G	A or G at 622- 103	intron 4	sequence variation
622- 116A/G	A or G at 622- 116	intron 4	sequence variation
622- 152G/C	G or C at 622- 152	intron 4	sequence variation
622- 16 T/C	622 - 16 T>C	intron 4	sequence variation
622- 1G- >A	G to A at 622- 1	intron 4	mRNA splicing defect
622- 2A- >C	A to C at 622- 2	intron 4	mRNA splicing defect
622- 2A- >G	A to G at 622- 2	intron 4	mRNA splicing defect
624delT	deletion of T at 624	5	frameshift
650delATAAA	Deletion of ATAAA	5	Frameshift
	at 650
657delA	deletion of A at 657	5	frameshift
663delT	deletion of T at 663	5	frameshift
675del4	deletion of TAGT	5	frameshift
	from 675
676A/G	A or G at 676	5	sequence variation
681delC	deletion of C at 681	5	frameshift
710_711+ 5de17	Deletion of	5
	AAGTATG between
	710 and 711+ 5
711+ 1G- >T	G to T at 711+ 1	intron 5	mRNA splicing defect
711+ 34A- >G	A to G at 711+ 34	intron 5	mRNA splicing defect
711+ 3A- >C	A to C at 711+ 3	intron 5	mRNA splicing defect
711+ 3A- >G	A to G at 711+ 3	intron 5	mRNA splicing defect
711+ 3A- >T	A to T at 711+ 3	intron 5	mRNA splicing defect
711+ 5G- >A	G to A at 711+ 5	intron 5	mRNA splicing defect
712- 1G- >T	G to T at 712- 1	intron 5	mRNA splicing defect
712- 92T/A	T or A at 712- 92	intron 5	sequence variation
733delG	Deletion of G at 733	6a	Frameshift
741C/T	C or T at 741	6a	sequence variation
- 741T- >G	T to G at - 741	promotor	promoter mutation
759A/G (A209A))	A or G at 759	6a	sequence variation
			(Ala at 209 no change)
- 790T9/8	9T or 8T at - 790	promotor	sequence variation
795G/A	G to A at 795	6a	Sequence variation. No
			change
- 816C- >T	C to T at - 816	promotor	promoter mutation
- 816delCTC	deletion of CTC	promotor	sequence variation
	at - 816
- 834T/G	T or G at - 834	promotor	sequence variation
852del22	deletion of 22 bp	6a	frameshift
	from 852
873C/T	C or T at 873	6a	sequence variation
874Ins TACA	Insertion of 4 bp	6a	stop codon at amino
	(TACA) at 874		acid 257 in exon 6b
875+ 1G- >A	G to A at 875+ 1	intron 6a	mRNA splicing defect
875+ 1G- >C	G to C at 875+ 1	intron 6a	mRNA splicing defect
875+ 40A/G	A or G at 875+ 40	intron 6a	sequence variation
876- 10del8	deletion of 8 bp	intron 6a	mRNA splicing defect
	from 876- 10
876- 14del12	deletion of 12 bp	intron 6a	mRNA splicing defect
	from 876- 14
876- 3C- >T	C to T at 876- 3	intron 6a	splicing mutation
876- 8 A- >C	876 - 8 A>C	intron 6a	mRNA splicing defect
- 895T/G	T or G at - 895	promotor	sequence variation
	upstream of the cap
	site
905delG	deletion of G	6b	frameshift
	at 905
- 912dupT	deletion of T at	promotor	Sequence variation
	nucleotide - 912
935delA	deletion of A	6b	frameshift
	at 935
936delTA	deletion of TA	6b	frameshift
	from 936
- 94G- >T	G to T at - 94	promotor	promoter mutation
977insA	insertion of A	6b	frameshift
	after 977
989- 992insA	Insertion of A	6b	Frameshift
	at 989- 992
991del5	deletion AACTT	6b	frameshift
	from 991 or
	CTTAA from 993
994del9	deletion of	6b	mRNA splicing defect
	TTAAGACAG from 994
99C/T	C or T at 99	promotor	sequence variation
A1006E	C to A at 3149	17a	Ala to Glu at 1006
A1009T	G to A at 3157	17a	Ala to Thr at 1009
A1025D	C to A at 3206	17a	Substitution of alanine
			to aspartic acid at
			position 1025
A1067D	C to A at 3332	17b	Ala to Asp at 1067
A1067G	C to G at 3332	17b	Ala to Gly at 1067
A1067P	G to C at 3331	17b	Ala en Pro at 1067
A1067T	G to A at 3331	17b	Ala to Thr at 1067
A1067V	C to T at 3332	17b	Ala to Val at 1067
A107G	C to G at 452	4	Ala to Gly at 107
A1081P	G to C at 3373	17b	Ala to Pro at 1081
A1087P	G to C at 3391	17b	Ala to Pro at AS 1087
A1136T	G to A at 3538	18	Ala to Thr at 1136
A120T	G to A at 490	4	Ala to Thr at 120
A120V	C to T at 491	4	Ala to Val at 120
A1319E	C to A at 4088	21	Ala to Glu at 1319
A1364V	C to T at 4223	22	Ala to Val at 1364 CBAVD
A141D	C to A at 554	4	Ala to Asp at 141
A155P	G to C at 595	4	Ala to Pro at 155
A198P	G to C at 724	6a	Ala to Pro at 198
A209S	G to T at 757	6a	Ala to Ser at 209
A238V	C to T at 845	6a	Ala to Val at 238
A299T	G to A at 1027	7	Ala to Thr at 299
A309A (1059C/G)	C or G at 1059	7	sequence variation
A309D	C to A at 1058	7	Ala to Asp at 309
A309G	C to G at 1058	7	Ala to Gly at 309
A309T	G to A at 1057	7	Ala to Thr at 309
A309V	C to T at 1058	7	Ala to Val at 309
A349V	C to T at 1178	7	Ala to Val at 349
A399D	C to A at 1328	8	Ala to Asp at 399
A399V	C to T at 1328	8	Ala to Val at 399
A455E	C to A at 1496	9	Ala to Glu at 455
A46D	C to A at 269	2	Ala to Asp at 46
A534E	C to A at 1733	11	Ala to Glu at 534
A559E	C to A at 1808	11	Ala to Glu at 559
A559T	G to A at 1807	11	Ala to Thr at 559
A559V	C to T at 1808	11	Ala to Val at 559
A561E	C to A at 1814	12	Ala to Glu at 561
A566T	G to A at 1828	12	Ala to Thr at 566
A613T	G to A at 1969	13	Ala to Thr at 613
A72D	C to A at 347	3	Ala to Asp at 72
A72T	G to A at 346	3	Ala to Thr at 72
A800G	C to G at 2531	13	Ala to Gly at 800
A959V	C to T at 3008	15	Ala to Val at 959
A96E	C to A at 419	4	Ala to Glu at 96
C225R	T to C at 805	6a	Cys to Arg at 225
C225X	T to A at 807	6a	Cys to Stop at 225
C276X	C to A at 960	6b	Cys to Stop at 276
C491R	T to C at 1603	10	Cys to Arg at 491
C524X	C to A at 1704	10	Cys to Stop at 524
C866R	T to G at 2728	14a	Cys to Arg at 866
C866S	T to A at 2728	14a	Cys to Ser at 866
C866Y	G to A at 2729	14a	Cys to Tyr at 866
CF25kbdel	Complex deletion/	intron 3
	rearrangement
CFTR40kbdel	deletion of exons	4, 5, 6a,	large deletion from
	4- 10	6b, 7, 8,	intron 3 to intron 10
		9, 10
CFTR40kbdel	deletion of exons	4, 5, 6a,	large deletion from
	4- 10	6b, 7, 8,	intron 3 to intron 10
		9, 10
CFTR50kbdel	complex deletion	4, 5, 6a,	complex deletion
	involving exons	6b, 7, 11,
	4- 7 and 11- 18	12, 13,
		14a, 14b,
		15, 16,
		17a, 17b,
		18
CFTRdele1	Deletion of exon 1	1	A small peptide of 17
	from nucleotide 136		residues if translation
	(codons 2- 18) to		starts at the same ATG
	intron 1 nucleotide +		or another protein
	69 and insertion of		(possibly CFTR-like)
	an inverted and		if another ATG is
	complementary sequence		choosen.
	of intron 1 (nucleotide
	185+ 4191 to + 4488)
	and addition of a G
	at the junction.
CFTRdele11- 16Ins35bp	Gross deletion of	11, 12,	The in-frame deletion
	47.5 kb going from	13, 14a,	of exons 11 to 16 was
	IVS10+ 12 to IVS16+		predicted to result in
	403 that removed	14b, 15,	a protein lacking amino
	exons 11 to 16	16	acids 529 to 996; this
	inclusive, together		includes the carboxy
	with an		terminal end of NBD1,
	insertion of 35 bp		the entire regulatory R
			domain and transmembrane-
			spanning regions TM7
			and TM8.
CFTRdele1- 24	deletion of the	1, 2, 3, 4,	absence of CFTR expression.
	whole CFTR gene	5, 6a, 6b,
		7, 8, 9,
		10, 11,
		12, 13,
		14a, 14b,
		15, 16,
		17a, 17b,
		18, 19,
		20, 21,
		22, 23,
		24
CFTRdele14a	deletion of >=1.2 kb	14a	aberrant mRNA splicing
	including exon 14a
CFTRdele14b- 17b	9890 bp deletion	14b, 15,	Removes 5 coding exons
		16, 17a,
		17b
CFTRdele14b- 18	deletion of 20 kb from	14b, 15,	deletion of amino acids 874-
	exons 14b through 18	16, 17a,	1156
		17b, 18
CFTR- dele 16- 17a- 17b	3040+ 1085_3499+	16, 17a,	Large in frame deletion
	260del7201	17b	removing exons 16,
			17a, 17b
CFTRdele16- 17b	deletion of 7 kb	16, 17a,	large deletion from
	starting at intron 15	17b	intron 15 to intron 17b
CFTRdele17b18	deletion of exons	17b, 18	frameshift
	17b and 18
CFTRdele19	deletion of 5.3 kb,	19
	removing exon 19
CFTRdele1Ins299bp	This indel involved	1
	the deletion of
	119 bp extending from
	coding position 4 (A
	of the ATG- translation
	initiation codon being
	defined as 1) to IVS1+ 69
	that removed nearly the
	entire coding sequence
	of exon 1, and the
	insertion of 299 bp
	at the deletion junction
CFTRdele1 or	136_185+	1, intron	Deletion of exon 1 from
136del119ins299	69del119bpins299bp	1	nucleotide 136 (codons
			2-18) to intron 1
			nucleotide +69 and
			insertion of an inverted and
			complementary sequence of
			intron 1 (nucleotide 185+ 4191
			to +4488) and addition of a G
			at the junction. A small
			peptide of 17 residues
			if translation starts
			at the same ATG or
			another protein (possibly
			CFTR-like) if another ATG is
			choosen.
CFTRdele2	deletion of exon 2	2	frameshift
CFTR- dele2	186- 1161_296+	2	Large in frame deletion
	1603del2875		removing exon2
CFTRdele21	deletion of exon 21	21	large deletion from exon 21
CFTRdele2- 10	deletion of 95.7 kb	2, 3, 4, 5,	frameshift
	starting in intron 1	6a, 6b, 7,
		8, 9, 10
CFTRdele22, 23	This deletion extends	22, 23	The loss of exons 22 and 23
	from nucleotide -		was in-frame and was
	78 of intron		predicted to result in a CFTR
	21 (the end of intron		protein lacking amino acids
	21 being defined as -		1322 to 1414; this constitutes
	1) to nucleotide +		the carboxy terminal end of
	577 of intron 23		the newly defined nucleotide-
	(the beginning of		binding domain (NBD) 2 of the
	intron 23 being defined		protein
	as + 1) with a loss
	of 1532 nucelotides
CFTRdele2, 3	deletion of exons	2, 3	frameshift
	2 and 3
CFTRdele3- 10, 14b- 16	Complex deletion	3, 4, 5,	Complex deletion
	involving exons	6a, 6b, 7,
	3- 10 and 14b- 16	8, 9, 10,
		14b, 15,
		16
CFTRdele3- 10, 14b- 16	Complex deletion	3, 4, 5,	Complex deletion
	involving exons	6a, 6b, 7,
	3- 10 and 14b- 16	8, 9, 10,
		14b, 15,
		16
CFTRdele4- 6aIns6bp	Deletion of 18, 654	4, 5, 6a	This large deletion disrupted
	bp encompassing exons		the reading frame of the
	4, 5, and 6a,		protein
	together with an
	insertion of 6 bp
CFTRdele4Ins41bp	Gross deletion	4	This deletion was in-frame and
	of 8, 165 bp		was predicted to lead to the
	spanning exon 4,		synthesis of a protein lacking
	together with an		amino acids 92-163, a stretch
	insertion of 41 bp		that includes a part of TM1
			and the the entire TM2
CFTRdup10_18	Duplication of	10, 11,	The position and orientation of
	exons 10 to 18	12, 13,	the duplicated region have not
		14a, 14b,	been determined. However,
		15, 16,	given the classical CF
		17a, 17b,	phenotype, it is hypothesized
		18	that it is located inside the
			CFTR gene.
CFTRdup11_13	Duplication of	11, 12,	The position and orientation of
	exons 11 to 13	13	the duplicated region have not
			been determined.
CFTRdup1-3	Duplication of	1, 2, 3	Large rearrangement. The
	exons 1 to 3		break points and orientation
			are being assessed
CFTRdup4-8	Duplication of	4, 5, 6, 7,	Complex rearrangement. The
	exons 4 to 8	8	position and orientation of the
			duplicated region is not
			determined so far. However,
			given the classical CF
			phenotype, it is hypothesized
			that it is located inside the
			CFTR gene.
Complex repeats		intron	sequence variation
		17b
D110E	C to A at 462	4	Asp to Glu at 110
D110H	G to C at 460	4	Asp to His at 110
D110N	G to A at 460	4	Asp to Asn at 110
D110Y	G to T at 460	4	Asp to Tyr at 110
D1152H	G to C at 3586	18	Asp to His at 1152
D1154G	A to G at 3593	18	Asp to Gly at 1154 (CBAVD)
D1154Y	G to T at 3592	18	Asp to Tyr at 1154
D1168G	A to G at 3635	19	Asp to Gly at 1168
D1270N	G to A at 3940	20	Asp to Asn at 1270
D1270Y	G to T at 3940	20	Asp to Tyr at 1270
D1305E	T to A at 4047	21	Asp to Glu at 1305
D1312G	A to G at 4067	21	Asp to Gly at 1312
D1377H	G to C at 4261	22	Asp to His at 1377
D1445N	G to A at 4465	24	Asp to Asn at 1445
D192G	A to G at 707	5	Asp to Gly at 192
D192N	G to A at 706	5	Asp to Asn at 192
D36N	G to A at 238	2	Asp to Asn at 36
D373E	T to G at 1251	8	Asp to Glu a 373
D443Y	G to T at 1459	9	Asp to Tyr at 443
D44G	A to G at 263	2	Asp to Gly at 44
D513G	A to G at 1670	10	Asp to Gly at 513 (CBAVD)
D529G	A to G at 1718	11	Asp to Gly at 529
D529H	G to C at 1717	11	Asp to His at 529
D537E	C to A or C to	11	Asp to Glu at 537
	G at 1743
D565G	A to G at 1826	12	Asp to Gly at 565
D572N	G to A at 1846	12	Asp to Asn at 572
D579A	A to C at 1868	12	Asp to Ala at 579
D579G	A to G at 1868	12	Asp to Gly at 579
D579Y	G to T at 1867	12	Asp to Tyr at 579
D585	A to G at 305	3	Asp to Gly at 58
D58N	G to A at 304	3	Asp to Asn at 58
D614G	A to G at 1973	13	Asp to Gly at 614
D614Y	G to T at 1972	13	Asp to Tyr at 614
D639Y	G to T at 2047	13	Asp to Tyr at 639
D651H	G to C at 2083	13	Asp to His at 651
D651N	G to A at 2083	13	Asp to Asn at 651
D674V	A to T at 2153	13	Asp to Val at 674
D806G	A to G at 2549	13	Asp to Gly at 806
D828G	A to G at 2615	13	Asp to Gly at 828
D836Y	G to T at 2638	14a	Asp to Tyr at 836
D891G	A to G at 2804	15	Asp to Gly at 891
D924N	G to A at 2902	15	Asp to Asn at 924
D979A	A to C at 3068	16	Asp to Ala at 979 (CBAVD)
D979V	A to T at 3068	16	Asp to Val at 979
D985H	G to C at 3085	16	Asp to His at 985
D985Y	G to T at 3085	16	Asp to Tyr at 985
D993G	A to G at 3110	16	Asp to Gly at 993
D993Y	G to T at 3109	16	Asp to Tyr at 993
delePr- 3	Large deletion	promoter,
		1, 2, 3
delEx2- 6b	185+ 2909_1002-	2, intron	The deletion of exons 2 to 6b
	1620del55429ins17	2, 3,	is in frame and would lead to
	((insertion of	intron 3,	remove 272 residues.
	GTACTCAACAGCTCTAG	4, intron
	(SEQ ID NO: 11))	4, 5,
		intron 5,
		6a, intron
		6a, 6b,
		intron 6b
delEx2- 9	c.53+ 9?711_1392+	1, intron	Large deletion of exons 2-9
	2?670del61?634	1,2,	(intron 1 to intron 9), out of
		intron 2,	frame
		3, intron
		3, 4,
		intron 4,
		5, intron
		5, 6a,
		intron 6a,
		6b, intron
		6b, 7,
		intron 7,
		8, intron
		8, 9,
		intron 9
Del exon 17a- 17b	Deletion of exons	17a, 17b	Truncation of CFTR protein in
	17a- 17b		TM2.
Del exon 17a- 17b- 18	Deletion of exons	17a, 17b,	in-frame deletion, joining of
	17a- 18	18	exons 16 to 19; deletion of
			terminal domain of TM2.
Del exon 22- 23	Deletion of exons	22, 23	In-frame deletion that is
	22- 23		predicted to remove the
			terminal part of NBD2
Del exon 22- 24	Deletion of Exons	22, 23,	Predicted Removal of terminal
	22, 23, 24	24	portion of CFTR protein
Del exon 2- 3	Deletion of exons	2, 3	Predicted truncation of the
	2, 3		CFTR Protein
Del exon 4- 6a	Deletion of exons	4, 5, 6a	Predicted truncation of the
	4, 5, 6a		CFTR protein in TM1.
Del Pr- Ex1	Deletion of Promoter,	promotor,	Predicted Removal of CFTR
	Exon 1	1	gene expression and ATG
			start Codon.
Del Pr- Ex1- Ex2	Deletion of Promoter,	promotor,	Predicted Removal of CFTR
	Exon 1, Exon 2	1, 2	gene expression and ATG
			start Codon.
[delta]D192	deletion of TGA or	5	deletion of Asp at 192
	GAT from 706 or 707
[delta]E115	3 bp deletion of	4	deletion of Glu at 115
	475- 477
[delta]F311	deletion of 3 bp	7	deletion of Phe 310, 311 or
	between 1059 and		312
	1069
[delta]F508	deletion of 3 bp	10	deletion of Phe at 508
	between 1652 and
	1655
[delta]I507	deletion of 3 bp	10	deletion of Ile 506 or Ile 507
	between 1648 and
	1653
[delta]L1260	deletion of ACT	20	deletion of Leu at 1260 or
	from either 3909		1261
	or 3912
[delta]L453	deletion of 3 bp	9	deletion of Leu at 452 or 454
	between 1488 and
	1494
[delta]M1140	deletion of 3 bp	18	deletion of Met at 1140
	between 3550 and
	3553
[delta]T339	deletion of 3 bp	7	deletion of Thr at 1140
	between 1148 and
	1150
dup1716+ 51- >61	duplication of 11	intron 10	sequence variation
	bp at 1716+ 51
Dup ex 6b- 10 (gIVS6a+	A duplication of	6b, 7, 8,	Out-of-frame fusion of exon 10
415_IVS10+	exons 6b- 10. The	9, 10	to exon 6b
2987Dup26817bp)	duplication
	is 26817 bp long.
E1104X	G to T at 3442	17b	Glu to Stop at 1104
E1123del	Deletion of AAG	18	deletion of Glu at 1123
	at 3503 - 3505
E116K	G to A at 478	4	Glu to Lys at 116
E116Q	G to C at 478	4	Glu to Gln at 116
E1228G	A to G at 3815	19	Glu to Gly at 1228
E1308X	G to T at 4054	21	Glu to Stop at 1308
E1321Q	G to C at 4093	21	Glu to Gln at 1321
E1371X	G to T at 4243	22	Glu to Stop at 1371
E1401G	A to G at 4334	23	Glu to Gly at 1401
E1401K	G to A at 4333	23	Glu to Lys at 1401
E1401X	G to T at 4333	23	Glu to Stop at 1401
E1409K	G to A at 4357	23	Glu to Lys at 1409
E1409V	A to T at 4358	23	Glu to Val at 1409
E1418X	G to T at 4384	24	Glu to Stop at 1418
	(GAG->TAG)
E1473X	G to T at 4549	24	Glu to Stop at 1473
E193K	G to A at 709	5	Glu to Lys at 193
E193X	G to T at 709	5	Glu to Stop at 193
E217G	A to G at 782	6a	Glu to Gly at 217
E278del	deletion of AAG	6b	deletion of Glu at 278
	from 965
E279D	A to T at 969	6b	Glu to Asp at 279
E279D	A to T at 969	6b	Glu to Asp at 279
E292K	G to A at 1006	7	Glu to Lys at 292
E379K	G to A at 1267	8	Glu to Lys at 379
E379X	G to T at 1267	8	Glu to Stop at 379
E403D	G to C at 1341	8	Glu to Asp at 403
E407V	A to T at 1352	9	Glu to Val at 407
E474K	G to A at 1552	10	Glu to Lys at 474
E479X	G to T at 1567	10	Glu to Stop at 479
E504Q	G to C at 1642	10	Glu to Gln at 504
E504X	G to T at 1642	10	Glu to Stop at 504
E527G	A to G at 1712	10	Glu to Gly at 527
E527Q	G to C at 1711	10	Glu to Gln at 527
E528D	G to T at 1716	10	Glu to Asp at 528 (splice
			mutation)
E528K	G to A at 1714	10	Glu to Lys at 528
E56K	G to A at 298	3	Glu to Lys at 56
E585X	G to T at 1885	12	Glu to Stop at 585
E588V	A to T at 1895	12	Glu to Val at 588
E608G	A to G at 1955	13	Glu to Gly at 608
E60K	G to A at 310	3	Glu to Lys at 60
E60X	G to T at 310	3	Glu to Stop at 60
E656X	G to T at 2098	13	Glu to Stop at 656
E664X	G to T at 2122	13	Glu to Stop at 664
E672del	deletion of 3 bp	13	deletion of Glu at 672
	between 2145-2148
E692X	G to T at 2206	13	Glu to Stop at 692
E725K	G to A at 2305	13	Glu to Lys at 725
E730X	G to T at 2320	13	Glu to Stop at 730
E7X	G to T at 151	1	Glu to Stop at 7
E822K	G to A at 2596	13	Glu to Lys at 822
E822X	G to T at 2596	13	Glu to Stop at 822
E823X	G to T at 2599	13	Glu to Stop at 823
E826K	G to A at 2608	13	Glu to Lys at 826
E827X	G to T at 2611	13	Glu to Stop at 827
E831X	G to T at 2623	14a	Glu to Stop at 831
E92D	A to T at 408	4	Gly to Asp at 92
	(GAA->GAT)
E92K	G to A at 406	4	Glu to Lys at 92
E92X	G to T at 406	4	Glu to Stop at 92
F1016S	T to C at 3179	17a	Phe to Ser at 1016
F1052V	T to G at 3286	17b	Phe to Val at 1052
F1074L	T to A at 3354	17b	Phe to Leu at 1074
F1166C	T to G at 3629	19	Phe to Cys at 1166
F1257L	T to G at 3903	20	Phe to Leu at 1257
F1286S	T to C at 3989	20	Phe to Ser at 1286
F1300L	T to C at 4030	21	Phe to Leu at 1300
F1337V	T to G at 4141	22	Phe to Val at 1337 (CBAVD)
F200I	T to A at 730	6a	Phe to Ile at 200
F305V	T to G at 1045	7	Phe 305 Val
F311L	C to G at 1065	7	Phe to Leu at 311
F316L	T to G at 1080	7	Phe to Leu at 316
F508C	T to G at 1655	10	Phe to Cys at 508
F508S	T to C at 1655	10	Phe to Ser at 508
F587I	T to A at 1891	12	Phe to Ile at 587
F693L(CTT)	T to C at 2209	13	Phe to Leu at 693
F693L(TTG)	T to G at 2211	13	Phe to Leu at 693
F87L	T to C at 391	3	Phe to Leu at 87
F932S	T to C at 2927	15	Phe to Ser at 932
F994C	T to G at 3113	16	Phe to Cys at 994
G1003E	G to A at 3140	17a	Gly to Glu at 1003
G1003X	G to T at 3139	17a	Gly to Stop at 1003
G103X	G to T at 439	4	Gly to Stop at 103
G1047D	G to A at 3272	17b	Gly to Asp at 1047 and mRNA
			splicing defect (CBAVD)
G1047R	G to C at 3271	17a	Gly to Arg at 1047
G1061R	G to C at 3313	17b	Gly to Arg at 1061
G1069R	G to A at 3337	17b	Gly to Arg at 1069
G1123R	G to Cat 3499	17b	Gly to Arg at 1123 mRNA
			splicing defect
G1127E	G to A at 3512	18	Gly to Glu at 1127
G1130A	G to C at 3521	18	Gly to Ala at 1130
G1237S	G to A at 3841	19	Gly to Ser at 1237
G1244E	G to A at 3863	20	Gly to Glu at 1244
G1244R	G to A at 3862	20	Gly to Arg at 1244
G1244V	G to T at 3863	20	Gly to Val at 1244
G1247R(G->A)	G to A at 3871	20	Gly to Arg at 1247
G1247R(G->C)	G to C at 3871	20	Gly to Arg at 1247
G1249E	G to A at 3878	20	Gly to Glu at 1249
G1249R	G to A at 3877	20	Gly to Arg at 1249
G126D	G to A at 509	4	Gly to Asp at 126
G1349D	G to A at 4178	22	Gly to Asp at 1349
G1349S	G to A at 4177	22	Gly to Ser at 1349
G149R	G to A at 577	4	Gly to Arg at 149
G149V	G to T at 578	4	Gly to Val at 149
G178E	G to A at 665	5	Gly to Glu at 178
G178R	G to A at 664	5	Gly to Arg at 178
G194R	G to A at 712	6a	Gly to Arg at 194
G194V	G to T at 713	6a	Gly to Val at 194
G213V	G to T at 771	6a	Gly to Val at 213
G239R	G to A at 847	6a	Gly to Arg at 239
G241R	G to A at 853	6a	Gly to Arg at 241
G27E	G to A at 212	2	Gly to Glu at 27
G27R	G to A at 211	6b	Gly to Arg at 27
G27R(211G to C)	G to C at 211	2	Gly to Arg at 27
G27X	G to T at 211	2	Gly to Stop at 27
G314E	G to A at 1073	7	Gly to Glu at 314
G314R	G to C at 1072	7	Gly to Arg at 314
G314V	G to T at 1073	7	Gly to Val at 314
G330X	G to T at 1120	7	Gly to Stop at 330
G424S	G to A at 1402	9	Gly to Ser at 424
G458V	G to T at 1505	9	Gly to Val at 458
G480C	G to T at 1570	10	Gly to Cys at 480
G480D	G to A at 1571	10	Gly to Asp at 480
G480S	G to A at 1570	10	Gly to Ser at 480
G486X	G to T at 1588	10	Gly to Stop at 486
G542X	G to T at 1756	11	Gly to Stop at 542
G544S	G to A at 1762	11	Gly to Ser at 544
G544V	G to T at 1763	11	Gly to Val at 544 (CBAVD)
G550R	G to A at 1780	11	Gly to Arg at 550
G550X	G to T at 1780	11	Gly to Stop at 550
G551D	G to A at 1784	11	Gly to Asp at 551
G551S	G to A at 1783	11	Gly to Ser at 551
G576A	G to C at 1859	12	Gly to Ala at 576 (CAVD)
G576X	G to T at 1858	12	Gly to Stop at 576
G622D	G to A at 1997	13	Gly to Asp at 622
			(oligospermia)
G628R(G->A)	G to A at 2014	13	Gly to Arg at 628
G628R(G->C)	G to C at 2014	13	Gly to Arg at 628
G673X	G to T at 2149	13	Gly to Stop at 673
G723V	G to T at 2300	13	Gly to Val at 723
G745X(Gly745X)	G to T at 2365	13	Non-sense mutation
G85E	G to A at 386	3	Gly to Glu at 85
G85V	G to T at 386	3	Gly to Val at 85
G91R	G to A at 403	3	Gly to Arg at 91
G970D	G to A at 3041	16	Gly to Asp at 970
G970R	G to C at 3040	15	Gly to Arg at 970
G970S	G to A at 3040	15	Gly to Ser at 970
H1054D	C to G at 3292	17b	His to Asp at 1054
H1054L	A to T at 3293	17b	His to Leu at 1054
H1054R	A to G at 3293	17b	His to Arg at 1054
H1079P	A to C at 3368	17b	His to Pro at 1079
H1085R	A to G at 3386	17b	His to Arg at 1085
H1375P	A to C at 4256	22	His to Pro at 1375
H139L	A to T at 548	4	His to Leu at 139
H139R	A to G at 548	4	His to Arg at 139
H146R	A to G at 569	4	His to Arg at 146 (CBAVD)
H199Q	T to G at 729	6a	His to Gln at 199
H199R	A to G at 728	6a	His to Arg at 199
H199Y	C to T at 727	6a	His to Tyr at 199
H484R	A to G at 1583	10	His to Arg at 484
H484Y	C to T at 1582	10	His to Tyr at 484 (CBAVD)
H609L	A to T at 1958	13	His to Leu at 609
H609R	A to G at 1958	13	His to Arg at 609
H620P	A to C at 1991	13	His to Pro at 620
H620Q	T to G at 1992	13	His to Gln at 620
H939D	C to G at 2947	15	His to Asp at 939
H939R	A to G at 2948	15	His to Arg at 939
H949L	A to T at 2978	15	His to Leu at 949
H949R	A to G at 2978	15	His to Are at 949
H949Y	C to T at 2977	15	His to Tyr at 949
I1005R	T to G at 3146	17a	Ile to Arg at 1005
I1027T	T to C at 3212	17a	Ile to Thr at 1027
I1051V	A to G at 3283	17b	Ile to Val at 1051
I105N	T to A at 446	4	Ile to Asn at 105
I1139V	A to G at 3547	18	Ile to Val at 1139
I119V	A to G at 487	4	Iso to Val at 119
I1230T	T to C at 3821	19	Ile to Thr at 1230
I1234L	A to C at 3832	19	sequence variation
I1234V	A to G at 3832	19	Ile to Val at 1234
I125T	T to C at 506	4	Ile to Thr at 125
I1269N	T to A at 3938	20	Ile to Asn at 1269
I1328T	T to C at 4115	22	Ile to Thr at 1328
I132M	T to G at 528	4	Ile to Met at 132 (sequence
			variation)
I1366T	T to C at 4229	22	Ile to Thr at 1366
I1398S	T to G at 4325	23	Ile to Ser at 1398
I148N	T to A at 575	4	Ile to Asn at 148
I148T	T to C at 575	4	Ile to Thr at 148
I175V	A to G at 655	5	Ile to Val at 175
I177T	T to C at 662	5	Ile to Thr at 177
I203M	C to G at 741	6a	Ile to Met at 203
I285F	A to T at 985	6b	Ile to Phe at 285
I331N	T to A at 1124	7	Ile to Asn at 331
I336K	T to A at 1139	7	Ile to Lys at 336
I340N	T to A at 1151	7	Ile to Asn at 340
I444S	T to G at 1463	9	Ile to Ser at 444
I444T	T to C at 1463	9	Ile to Thr at 444
I497V	A to G at 1621	10	Ile to Val at 497
I502N	T to A at 1637	10	Ile to Asn at 502
I502T	T to C at 1637	10	Ile to Thr at 502
I506L	A to C at 1648	10	Ile to Leu at 506
I506S	T to G at 1649	10	Ile to Ser at 506
I506T	T to C at 1649	10	Ile to Thr at 506
I506V (1648A/G)	A or G at 1648	10	Ile or Val at 506
I539T	T to C at 1748	11	Ile to Thr at 539
I556V	A to G at 1798	11	Ile to Val at 556 (mutation)
I586V	A to G at 1888	12	Ile to Val at 586
I601F	A to T at 1933	13	Ile to Phe at 601
I618T	T to C at 1985	13	Ile to Thr at 618
I752S	T to G at 2387	13	Ileu to Ser at 752
	(ATC->AGC)
I807M	A or G at 2553	13	sequence variation
I807V	A to G at 2551	13	Ile to Val at 807
I840T	T to C at 2651	14a	Ile to Thr at 840
I918M	T to G at 2886	15	Ile to Met at 918
I980K	T to A at 3071	16	Ile to Lys at 980
I980M	A to G at 3072	16	Ile to Met at 980
I991V	A to G at 3103	16	Ile to Val at 991
IVS14a+ 17del5	5 by deletion	intron	sequence variation
	between 2751+ 17	14a
	and 2751+ 24
K1060T	A to C at 3311	17b	Lys to Thr at 1060
K1080R	A to G at 3371	17b	Lys to Arg at 1080
K114X	A to T at 472	4	Lys to Stop at 114
K1177R	A to G at 3662	19	Lys to Arg at 1177
K1177X	A to T at 3661	19	Lys to Stop at 1177
			(premature termination)
K1302R	A to G at 4037	4	Lys to Arg at 1302
	(AAA->AGA)
K1351E	A to G at 4183	22	Lys to Glu at 1351 (CBAVD)
K14X	Ato T at 172	1	Lys to Stop at 14
K162E	Ato G at 616	4	Lys to Glu at 162
K166Q	A to G at 628	5	Lys to Gln at 166
K464N	G to Tat 1524	9	Lys to Asn at 464; mRNA
			splicing defect
K536X	A to T at 1738	11	Lys to Stop codon at 536
K598X	A to T at 1924	13	Lys to Stop at 598
K64E	A to G at 322	3	Lys to Glu at 64
K683R	A to G at 2180	13	Lys to Arg at 683
K688X	A to T at 2194	13	Lys to Stop at 688
K68E	A to G at 334	3	Lys to Glu at 68
K68N	A to T at 336	3	Lys to Asn at 68
K710X	A to T at 2260	13	Lys to Stop at 710
K716X	AA to GT at	13	Lys to Stop at 716
	2277 and 2278
K830X	A to T at 2620	13	Lys to Stop at 830
K946X	A to T at 2968	15	Lys to Stop at 946
L101S	T to C at 434	4	Leu to Ser at 101
L101X	T to G at 434	4	Leu to Stop at 101
L102P	T to C at 437	4	Leu to Pro at 102
L102R	T to G at 437	4	Leu to Arg at 102
L1059L (3309A/G)	A or G at 3309	17b	sequence variation
L1059X	T to G at 3308	17b	Leu to Stop at 1059
L1065F	C to T at 3325	17b	Leu to Phe at 1065
L1065P	T to C at 3326	17b	Leu to Pro at 1065
L1065R	T to G at 3326	17b	Leu to Arg at 1065
L1077P	T to C at 3362	17b	Leu to Pro at 1077
L1093P	T to C at 3410	17b	Leu to Pro at 1093
L1096R	T to G at 3419	17b	Leu to Arg at 1096
L1156F	G to T at 3600	18	Leu to Phe at 1156
L1227S	T to C at 3812	19	Leu to Ser at 1227
L1254X	T to G at 3893	20	Leu to Stop at 1254
L126OR	T to G at 3911	20	Leu to Arg at 1260
L127X	T to G at 512	4	Leu to Stop at 127
L130V	C to G at 520	4	Leucine to Valine at 130
L1324P	T to C at 4103	22	Leu to Pro at 1324
L1335F	C to T at 4135	22	Leu to Phe at 1335
L1335P	T to C at 4136	22	Leu to Pro at 1335
L1339F	C to T at 4147	22	Leu to Phe at 1339
L137H	T to A at 542	4	Leu to His at 137
L137P	T to C at 542	4	Leu to Pro at 137 (sequence
			variation)
L137R	T to G at 542	4	Leu to Arg at 137
L1388Q	T to A at 4295	23	Leu to Gln at 1388 (CBAVD)
L1388V	C to G at 4294	23	Leu to Val at 1388
L138ins	insertion of CTA,	4	insertion of leucine at 138
	TAC or ACT at
	nucleotide 544,
	545 or 546
L1414S	T to C at 4373	23	Leu to Ser at 1414
L145H	T to A at 566	4	Leu to His at 145
L1480P	T to C at 4571	24	Leu to Pro a 1480
L159S	T to C at 608	4	Leu to Ser at 159
L159X	T to A at 608	4	Leu to Stop at 159
L15P	T to C at 176	1	Leu to Pro at 15
L165S	T to C at 626	5	Leu to Ser at 165
L1831	C to A at 679	5	Leu to Ile at 183
L206F	G to T at 750	6a	Leu to Phe at 206
L206W	T to G at 749	6a	Leu to Trp at 206
L210P	T to C at 761	6a	Leu to Pro at 210
L218X	T to A at 785	6a	Leu to Stop at 218
L227R	T to G at 812	6a	Leu to Arg at 227
L24F	G to C at 204	2	Leu to Phe at 24
L293M	C to A at 1009	7	Leu to Met at 293
L320F	A to T at 1092	7	Leu to Phe at 320
L320V	T to G at 1090	7	Leu to Val at 320 CAVD
L320X	T to A at 1091	7	Leu to Stop at 320
L327R	T to G at 1112	7	Leu to Arg at 327
L346P	T to C at 1169	7	Leu to Pro at 346
L365P	T to C at 1226	7	Leu to Pro at 365
L375F	A to C at 1257	8	Leu to Phe at 375 (CUAVD)
L383L (1281G/A)	G or A at 1281	8	sequence variation
L383S	T to C at 1280	8	Leu to Ser at 383
L468P	T to C at 1535	10	Leu to Pro at 468
L548Q	T to A at 1775	11	Leu to Gln at 548
L558S	T to C at 1805	11	Leu to Ser at 558
L568F	G to T at 1836	12	Leu to Phe at 568 (CBAVD)
L568X	T to A at 1835	12	Leu to Stop at 568
L571S	T to C at 1844	12	Leu to Ser at 571
L594P	T to C at 1913	13	Leu to Pro at 594
L610S	T to C at 1961	13	Leu to Ser at 610
L619S	T to C at 1988	13	Leu to Ser at 619
L61P	T to C at 314	3	Leucine to Proline at position
			61
L633I	C to A at 2029	13	Leu to Ile at 633
L633P	T to C at 2030	13	Leu to Pro at 633
L636P	T to C at 2039	13	Leu to Pro at 636
L719X	T to A at 2288	13	Leu to Stop at 719
L732X	T to G at 2327	13	Leu to Stop at 732
L829L (2619A/G)	A or G at 2619	13	sequence variation
L867X	T to A at 2732	14a	Leu to Stop at 867
L88S	T to C at 395	3	Leu to Ser at 88
L88X(T->A)	T to A at 395	3	Leu to Stop at 88
L88X(T->G)	T to G at 395	3	Leu to Stop at 88
L90S	T to C at 401	3	Leu to Ser at 90
L927P	T to C at 2912	15	Leu to Pro at 927
L967S	T to C at 3032	15	Leu to Ser at 967
			(oligospermia)
L973F	TC to AT at	16	Leu to Phe at 973 (BAVD)
	3048 and 3049
L973H	T to A at 3050	16	Leu to His at 973
L973P	T to C at 3050	16	Leu to Pro at 973
L997F	G or C at 3123	17a	Leu or Phe at 997 (sequence
			variation)
M1028I	G to T at 3216	17a	Met to Ile at 1028
M1028R	T to G at 3215	17a	Met to Arg at 1028
M1101K	T to A at 3434	17b	Met to Lys at 1101
M1101R	T to G at 3434	17b	Met to Arg at 1101
M1105R	T to G at 3446	17b	Met to Arg at 1105
M1137R	T to G at 3542	18	Met to Arg at 1137
M1137T	T to C at 3542	18	Met to Thr at 1137
M1137V	A to G at 3541	18	Met to Val at 1137
M1140K	T to A at 3551	18	Met to Lys at 1140
M1210I	G to A at 3762	19	Met to Ile at 1210
M1210K	T to A at 3761	19	Met to Lys at 1210
M1407T	T to C at 4352	23	Met to Thr at 1407
M152L	A to T at 586	4	Met to Leu at 152
M152R	T to G at 587	4	Met to Arg at 152
M152V	A to G at 586	4	Met to Val at 152 (mutation)
M1I(ATA)	G to A at 135	1	no translation initiation
M1I(ATT)	G to T at 135	1	no translation initiation
M1K	T to A at 134	1	no translation initiation
M1L	A to C at 133	1	Met to Leu at 1
M1T	T to C at 134	1	Met to Thr at 1
M1V	A to G at 133	1	no translation initiation
M243L	A to C at 859	6a	Met to Leu at 243 (ATG to
			CTG)
M244K	T to A at 863	6a	Met to Lys at 244
M265R	T to G at 926	6b	Met to Arg at 265
M281T	T to C at 974	6b	Met to Thr at 281
M348K	T to A at 1175	7	Met to Lys at 348
M348T	T to C at 1175	7	Met to Thr at 348
M348V	A to G at 1174	7	Met to Val at AS 348
M394R	T to G at 1313	8	Met to Arg at 394
M469V	A to G 1537	10	Met to Val at 469
M470V	A or G at 1540	10	sequence variation
M498I	G to C at 1626	10	Met (ATG) to Ileu (ATC) at
			498
M595I	G to A at 1917	13	Met to Ile at 595
M595T	T to C at 1916	13	Met to Thr at 595
M82V	A to G at 376	3	Met to Val at 82
M952I	G to C at 2988	15	Met to Ile at 952 CBAVD
			mutation
M952T	T to C at 2987	15	Met to Thr at 952
M961I	G to T at 3015	15	Met to Ile at 961
N1088D	A to G at 3394	17b	Asn to Asp at 1088
N113I	A to T at 470	4	Asn to Ile
N1148K	C to A at 3576	18	Asn to Lys at 1148
N1148S	A to G at 3575	18	Asn to Ser at 1148
N1195T	A to C at 3716	19	Asn to Thr at 1195
N1303H	A to C at 4039	21	Asn to His at 1303
N1303I	A to T at 4040	21	Asn to Ile at 1303
N1303K	C to G at 4041	21	Asn to Lys at 1303
N1432K	C to G at 4428	24	sequence variation
N186K	C to A at 690	5	Asn to Lys at 186
N187K	C to A at 693	5	Asn to Lys at 187
N189K	C to A at 699	5	Asn to Lys at 189
N189S	A to G at 698	5	Asn to Ser at 189
N287Y	A to T at 991	6b	Asn to Tyr at 287
N369Y	A to T at 1318	8	Asn to Tyr at 396
N416S	A to G at 1379	9	Asn to Ser at 416
N418S	A to G at 1385	9	Asn to Ser at 418
N66S	A to G at 329	3	Asn to Ser at 66
N782K	C to A at 2478	13	Asn to Lys at 782
N900T	A to C at 2831	15	Asn to Thr at 900
P1013H	C to A at 3170	17a	Pro to His at 1013
P1013L	C to T at 3170	17a	Pro to Leu at 1013
P1021A	C to G at 3193	17a	Pro to Ala at 1021
P1021S	C to T at 3193	17a	Pro to Ser at 1021 (CBAVD)
P1072L	C to T at 3347	17b	Pro to Leu at 1072
P111A	C to G at 463	4	Pro to Ala at 111
P111L	C to T at 464	4	Pro to Leu at 111
P1290P (4002A/G)	A or G at 4002	20	sequence variation
P1290S	C to T at 4000	20	Pro to Ser at 1290
P1290T	C to A at 4000	20	Pro to Thr at 1290
P1306P (4050C/T)	C or T at 4050	21	sequence variation
P1372L	C to T at 4247	22	Pro to Leu at 1732
P1372T	C to A 4246	22	Pro to Thr at 1372
P140L	C to T at 551	4	Pro to Leu at 140
P140S	C to T at 550	4	Pro to Ser at 140
P205R	C to G at 746	6a	Pro to Arg at 205
P205S	C to T at 745	6a	Pro to Ser at 205
P324L	C to T at 1103	7	Pro to Leu at 324
P355S	C to T at 1195	7	Pro to Ser at 355
P439S	C to T at 1447	9	Pro to Ser at 439
P499A	C to G at 1627	10	Pro to Ala at 499 (CBAVD)
P574H	C to A at 1853	12	Pro to His at 574
P574S	C to T at 1852	12	Pro to Ser at 574
P5L	C to T at 146	1	Pro to Leu at 5
P67L	C to T at 332	3	Pro to Leu at 67
P750L	C to T at 2381	13	Pro to Leu at 750
P841R	C to G at 2654	14a	Pro to Arg at 841
P99L	C to T at 428	4	Pro to Leu at 99
poly-T tract	variable number	intron 8	sequence variation (3 variants
variations	(5T, 7T, 9T) of		of which IVS8-5T is affecting
	thymidines at the		splicing of exon 9)
	poly-T tract
	starting at
	position 1342-6
Q1035X	C to T at 3235	17a	Nonsense mutation
Q1042X	C to T at 3256	17a	Gln to Stop at 1042
Q1071H	G to T at 3345	17b	Gln to His at 1071
Q1071P	A to C at 3344	17b	Gln to Pro at 1071
Q1071X	C to T at 3343	17b	Gln to Stop at 1071
Q1100P	A to C at 3431	17b	Gln to Pro at 1100
Q1144X	C to T at 3562	18	Gln to Stop at 1144
Q1186Q (3690A/G)	A or G at 3690	19	sequence variation
Q1186X	C to T at 3688	19	Gln to Stop at 1186
Q1238R	A to G at 3845	19	Gln to Arg at 1238
Q1238X	C to T at 3844	19	Gln to Stop at 1238
Q1268R	A to G at 3935	20	Gln to Arg at 1268
Q1281X	C to T at 3973	20	Gln to Stop at 1281
Q1291H	G to C at 4005	20	Gln to His at 1291; mRNA
			splicing defect
Q1291R	A to G at 4004	20	Gln to Arg at 1291
Q1291X	C to T at 4003	20	Gln to Stop at 1291
Q1309H	G to T at 4059	21	Gln to His at 1309
Q1313K	C to A at 4069	21	Gln to Lys at 1313
Q1313X	C to T at 4069	21	Gln to Stop at 1313
Q1352E	C to G at 4186	22	Gln to Glu at 1352
Q1352H(G->C)	G to C at 4188	22	Gln to His at 1352
Q1352H(G->T)	G to T at 4188	22	Gln to His at 1352
Q1382X	C to T at 4276	23	Gln to Stop at 1382
Q1390X	4300C>T	23	Gln to stop at 1390
Q1411X	C to T at 4363	23	Gln to Stop at 1411
Q1412X	C to T at 4366	23	Gln to Stop at 1412
Q1463H	G to T at 4521	24	Gln to His a 1463
Q1476X	C to T at 4558	24	Gln to Stop at 1476
Q151K	C to A at 583	4	Gln to Lys at 151
	(CAG->AAG)
Q151X	C to T at 583	4	Gln to Stop at 151
Q179K	C to A at 667	5	Gln to Lys at 179
Q207X	C to T at 751	6a	Gln to Stop at 207
Q220R	A to G at 791	6a	Gln to Arg at 220
Q220X	C to T at 790	6a	Gln to Stop at 220
Q237E	C to G at 841	6a	Gln to Glu at 237
Q290X	C to T at 1000	6b	Gln to Stop at 290
Q2X (together with	C to T at 136 and	1	Gln to Stop at codon 2 and
R3W)	A to T at 139		Arg to Trp at codon 3
Q30X	C to T at 220	2	Gln to Stop at 30
Q353H	A to C at 1191	7	Gln to His at 353
Q353X	C to T at 1189	7	Gln to Stop at 353
Q359K/T360K	C to A at 1207 and	7	Glu to Lys at 359 and Thr to
	C to A at 1211		Lys at 360
Q359R	A to G at 1208	7	Gln to Arg at 359
Q378R	A to G at 1265	8	Gln to Arg at 378
Q39X	C to T at 247	2	Gln to Stop at 39
Q414X	C to T at 1372	9	Gln to Stop at 414
Q452P	A to C at 1487	9	Gln to Pro at 452
Q493P	A to C at 1610	10	Gln to Pro at 493
Q493R	A to G at 1610	10	Gln to Arg at 493
Q493X	C to T at 1609	10	Gln to Stop at 493
Q525X	C to T at 1705	10	Gln to Stop at 525
Q552K	C to A at 1786	11	Gln to Lys at 552
Q552X	C to T at 1786	11	Gln to Stop at 552
Q634X	C to T at 2032	13	Gln to Stop at 634
Q637X	C to T at 2041	13	Gln to Stop at 637
Q685X	C to T at 2185	13	Gln to Stop at 685
Q689X	C to T at 2197	13	Gln to Stop at 689
Q715X	C to T at 2275	13	Gln to Stop at 715
Q720X	C to T at 2290	13	Gln to Stop at 720
Q781X	C to T at 2473	13	Gln to Stop at 781
Q814X	C to T at 2572	13	Gln to Stop at 814
Q890R	A to G at 2801	15	Gln to Arg at 890
Q890X	C to T at 2800	15	Gln to Stop at 890
Q98P	A to C at 425	4	Gln to Pro at 98
Q98R	A to G at 425	4	Gln to Arg at 98
Q98X	C to T at 424	4	Gln to Stop at 98 (Pakistani
			specific)
R1048G	A to G at 3274	17b	Arg to Gly a 1048
R1066C	C to T at 3328	17b	Arg to Cys at 1066
R1066H	G to A at 3329	17b	Arg to His at 1066
R1066L	G to T at 3329	17b	Arg to Leu at 1066
R1066S	C to A at 3328	17b	Arg to Ser at 1066
R1070P	G to C at 3341	17b	Arg to Pro at 1070
R1070Q	G to A at 3341	17b	Arg to Gln at 1070
R1070W	C to T at 3340	17b	Arg to Trp at 1070
R1102X	A to T at 3436	17b	Arg to Stop at 1102
R1128X	A to T at 3514	18	Arg to Stop at 1128
R1158X	C to T at 3604	19	Arg to Stop at 1158
R1162X	C to T at 3616	19	Arg to Stop at 1162
R117C	C to T at 481	4	Arg to Cys at 117
R117G	C to G at 481	4	Arg to Gly at 117
R117H	G to A at 482	4	Arg to His at 117
R117L	G to T at 482	4	Arg to Leu at 117
R117P	G to C at 482	4	Arg to Pro at 117
R1239S	G to C at 3849	19	Arginine to Serine at 1239
R1283K	G to A at 3980	20	Arg to Lys at 1283
R1283M	G to T at 3980	20	Arg to Met at 1283
R1358S	A to T at 4206	22	Arg to Ser at 1358
R1422W	C to T at 4396	24	Arg to Trp at 1422
R1438W	C to T at 4444	24	Arg to Try at 1438
R1453W	C to T at 4489	24	Arg to Trp at 1453
R170C	C to T at 640	5	Arg to Cys at 170
R170G	C to G at 640	5	Arg to Gly at 170
R170H	G to A at 641	5	Arg to His at 170
R248T	G to C at 875	6a	Arg to Thr at 248 (CBAVD)
R258G	A to G at 904	6b	Arg to Gly at 258
R297Q	G to A at 1022	7	Arg to Gln at 297
R297W	C to T at 1021	7	Arg to Trp at 297
R31C	C to T at 223	2	Arg to Cys at 31
R31L	G to T at 224	2	Arg to Leu at 31
R334L	G to T at 1133	7	Arg to Leu at 334
R334Q	G to A at 1133	7	Arg to Gln at 334
R334W	C to T at 1132	7	Arg to Trp at 334
R347C	C to T at 1171	7	Arg to Cys at 347
R347H	G to A at 1172	7	Arg to His at 347
R347L	G to T at 1172	7	Arg to Leu at 347
R347P	G to C at 1172	7	Arg to Pro at 347
R352G	C to G at 1186	7	Arg to Gly at 352
R352Q	G to A at 1187	7	Arg to Gln at 352
R352W	C to T at 1186	7	Arg to Trp at 352
R516G	A to G at 1678	10	Arg to Gly at 516
R553G	C to G at 1789	11	Arg to Gly at 553
R553Q	G to A at 1790	11	Arg to Gln at 553 (associated
			with [delta]F508;
R553X	C to T at 1789	11	Arg to Stop at 553
R555G	A to G at 1795	11	Arg to Gly at 555
R55K	G to A at 296	2	Arg to Lys at 55
R560G	A to G at 1810	11	Ala to Gly at 560
R560K	G to A at 1811	11	Arg to Lys at 560
R560S	A to C at 1812	12	Arg to Ser at 560
R560T	G to C at 1811	11	Arg to Thr at 560; mRNA
			splicing defect
R600G	A to G at 1930	13	Arg to Gly at 600
R668C	C or T at 2134	13	sequence variation
R709Q	G to A at 2258	13	Arg to Gln at 709
R709X	C to T at 2257	13	Arg to Stop at 709
R735K	G to A at 2336	13	Arg to Lys at 735
R74Q	G to A at 353	3	Arg to Gln at 74
R74W	C to T at 352	3	Arg to Trp at 74
R751P	G to C at 2384	13	Arg to Pro at 751
R75L	G to T at 356	3	Arg to Leu at 75
R75Q	G or A at 356	3	sequence variation
R75X	C to T at 355	3	Arg to Stop at 75
R764X	C to T at 2422	13	Arg to Stop at 764
R766M	G to T at 2429	13	Arg to Met at 766
R785X	C to T at 2485	13	Arg to Stop at 785
R792G	C to G at 2506	13	Arg to Gly at 792
R792X	C to T at 2506	13	Arg to Stop at 792
R810G	A to G at 2560	13	Arg to Gly at 810
R851L	G to T at 2684	14a	Arg to Leu at 851
R851X	C to T at 2683	14a	Arg to Stop at 851
R933G	A to G at 2929	15	Arg to Gly at 933
R933S	A to T at 2931	15	Arg to Ser at 933 (CBAVD)
S108F	C to T at 455	4	Ser to Phe at 108
S10R	A to C at 160	1	Ser to Arg at 10
S1118C	C to G at 3485	17b	Ser to Cys at 1118
S1118F	C to T at 3485	17b	Ser to Phe at 1118
S1159F	C to T at 3608	19	Ser to Phe at 1159
S1159P	T to C at 3607	19	Ser to Pro at 1159
S1161R	A to C at 3613	19	Ser to Arg at 1161
	or C to G at 3615
S1196X	C to G at 3719	19	Ser to Stop at 1196
S1206X	C to G at 3749	19	Ser to Stop at 1206
S1206X(C>A)	C to A at 3749	19	Ser to Stop at 1206
S1235R	T to G at 3837	19	Ser to Arg at 1235
S1251N	G to A 3884	20	Ser to Asn at 1251
S1255L	C to T at 3896	20	Ser to Leu at 1255
S1255P	T to C at 3895	20	Ser to Pro at 1255
S1255X	C to A at 3896	20	Ser to Stop at 1255 and Ile to
	and A to G at		Val at 1203
	3739 in exon 19
S1311R	A to C at 4063	21	Ser to Arg at 1311
	or T to A
	or G at 4065
S13F	C to T at 170	1	Ser to Phe at 13
S1426F	C to T at 4409	24	Ser to Phe at 1426
S1426P	T to C at 4408	24	Ser to Pro at 1426
S1455X	C to G at 4496	24	Ser to Stop at 1455
S158N	G to A at 605	4	Ser to Asn at 158
S158R	A to C at 604	4	Ser to Arg at 158
S158T	G to C at 605	4	Ser to Thr at 158
S18G	A to G at 184	1	Ser to Gly at 18
S307N	G to A at 1052	7	Ser to Asn at 307
S313X	C to A at 1070	7	Ser to Stop
S321P	T to C at 1093	7	Ser to Pro at 321
S341P	T to C at 1153	7	Ser to Pro at 341
S364P	T to C at 1222	7	Ser to Pro at 364
S42F	C to T at 257	2	Ser to Phe at 42
S431G	A to G at 1423	9	Ser to Gly a 431
S434X	C to G at 1433	9	Ser to Stop at 434
S466L	C to T at 1529	10	Ser to Leu at 466 (CBAVD)
S466X(TAA)	C to A at 1529	10	Ser to Stop at 466
S466X(TAG)	C to G at 1529	10	Ser to Stop at 466
S485C	A to T at 1585	10	Ser to Cys at 485
S489X	C to A at 1598	10	Ser to Stop at 489
S492F	C to T at 1607	10	Ser to Phe at 492
S4X	C to A at 143	1	Ser to Stop at 4
S50P	T to C at 280	2	Ser to Pro at 50
S50Y	C to A at 281	2	Ser to Tyr at 50 (CBAVD)
S519G	A to G at 1687	10	Ser to Gly at 519
S549I	G to T at 1778	11	Ser to Ile at 549
S549N	G to A at 1778	11	Ser to Asn at 549
S549R(A->C)	A to C at 1777	11	Ser to Arg at 549
S549R(T->G)	T to G at 1779	11	Ser to Arg at 549
S573C	C to G at 1850	12	Ser to Cys at 573
S589I	G to T at 1898	12	Ser to Ile at 589 (splicing)
S589N	G to A at 1898	12	Ser to Asn at 589 (mRNA
			splicing defect)
S660T	T to A at 2110	13	Ser to Thr a 660
S686Y	C to A at 2189	13	Ser to Tyr at 686
S712C	C to G at 2267	13	Ser to Cys at 712
S737F	C to T at 2342	13	missense
S737F	C to T at 2342	13	Ser to Phe at 737
S753R	C to G at	13	Serine to arginine at 753
	position 2391
S776X	C to G at 2459	13	Ser to Stop at 776
S813P	T to C at 2569	13	Ser to Pro at 813
S895T	G to C at 2816	15	Ser to Thr at 895
S902R	C to G at 2838	15	Ser to Arg at 902
S911R	A to C at 2863	15	Ser to Arg at 911
	or T to A or
	T to G at 2865
S912L	C to T at 2867	15	Ser to Leu at 912
S912X	C to A at 2867	15	Ser to Stop at 912
S945L	C to T at 2966	15	Ser to Leu at 945
S977F	C to T at 3062	16	Ser to Phe at 977
S977P	T to C at 3061	16	Ser to Pro at 977
T1053I	C to T at 3290	17b	Thr to Ile at 1053 (CBAVD)
T1057A	A to G at 3301	17b	Thr to Ala at 1057
T1086A	A to G at 3388	17b	Thr to Ala at 1086
T1086I	C to T at 3389	17b	Thr to Ile at 1086
T1142I	C to T at 3557	18	Thr to Ile at 1142
T1246I	C to T at 3869	20	Thr to Ile at 1246 (mutation)
T1252P	A to C at 3886	20	Thr to Pro at 1252
T1263A	A to G at 3919	20	Thr to Ala at 1263
T1263I	C to T at 3920	20	Thr to Ile at 1263
T1299I	C to T at 4028	21	Thr to Ile at 1299
T338A	A to G at 1144	7	Thr to Ala at 338
T338I	C to T at 1145	7	Thr to Ile at 338
T351I	C to T at 1184	7	Thr to Ile at 351
T351S	C or G at 1184	7	sequence variation
T360R	C to G at 1211	7	sequence variation (Thr to Arg
			at 360)
T388M	C to T at 1295	8	Thr to Met at 388 (sequence
			variation)
T388X	AC to TA at 1294	8	Thr to Stop at 388
T501A	A to G at 1633	10	Thr to Ala at 501
T582I	C to T at 1877	12	Thr to Ile at 582
T582R	C to G at 1877	12	Thr to Arg at 582
T582S	A to T at 1876	12	Thr to Ser at 582
T599T (1929T/A)	T or A at 1929	13	sequence variation
T604I	C to T at 1943	13	Thr to Ile at 604
T604S	C to G at 1943	13	Thr to Ser at 604
T665S	A to T at 2125	13	Thr to Ser at 665
T760M	C to T at 2411	13	Thr to Met at 760
T788I	C to T at 2495	13	Thr to Ile at 788
T896I	C to T at 2819	15	Thr to Ile at 896
T908N	C to A at 2855	15	Thr to Asn at 908
TAAA repeats	9 or 11 repeats of TAAA	intron 9	sequence variation
	(SEQ. ID NO: 12) at
TTGA repeats	5-7 copies of repeat	intron 6a	sequence variation
	at around 876-31
V1008D	T to A at 3155	17a	Val to Asp at 1008
V1020E	T to A at 3191	17a	Val to Glu at 1020
V1108L	G to C at 3454	17b	Val to Leu at 1108
V1129G	3518T>G	18	Val to Gly at 1129
V1147I	G to A at 3571	18	Val to Ile at 1147
V1153E	T to A at 3590	18	Val to Glu at 1153 (CBAVD)
V1190D	T to A at 3701	19	Val to Asp at 1190
V1212I	G to A at 3766	19	Val to Ile at 1212
V1240G	T to G at 3851	20	Val to Gly at 1240
V1293I	G to A at 4009	21	Val to Ile at 1293
V1318A	T to C at 4085	21	Val to Ala at 1318
V1397E	T to A at 4322	23	Val to Glu at 1397
V201M	G to A at 733	6a	Val to Met at 201
V232D	T to A at 827	6a	Val to Asp at 232 (CBAVD)
V317A	T to C at 1082	7	Val to Ala at 317
V322A	T to C at 1097	7	Val to Ala at 322 (mutation)
V322M (1096(G/A))	G or A at 1096	7	sequence variation
V392A	T to C at 1307	8	Val to Ala at 392 CAVD
V392G	T to G at 1307	8	Val to Gly at 392
V456A	T to C at 1499	9	Val to Ala at 456 (sequence
			variation)
V456F	G to T at 1498	9	Val to Phe at 456
V520F	G to T at 1690	10	Val to Phe at 520
V520I	G to A at 1690	10	Val to Ile at 520
V562I	G to A at 1816	12	Val to Ile at 562
V562L	G to C at 1816	12	Val to Leu at 562
V603F	G to T at 1939	13	Val to Phe at 603
V754M	G to A at 2392	13	Val to Met at 754
V855I	G to A at 2695	14a	Val to Ile at 855 (sequence
			variation)
V920L	G to T at 2890	15	Val to Leu at 920
V920M	G to A at 2890	15	Val to Met at 920
V922L	G to C at 2896	15	Val to Leu at 922
V938G	T to G at 2945	15	Val to Gly at 938 (CAVD)
V938L	G to C at 2944	15	Val to Leu at 938
W1063X	G to A at 3321	17b	Trp to Stop at 1063
W1089X	G to A at 3398	17b	Trp to Stop at 1089
W1098L	G to T at 3425	17b	Trp to Leu at 1098
W1098R	T to C at 3424	17b	Trp to Arg at 1098
W1098X(TAG)	G to A at 3425	17b	Trp to Stop at 1098
W1098X(TGA)	G to A at 3426	17b	Trp to Stop at 1098
W1145X	G to A at 3567	18	Trp to Stop at 1154
W1204X(3743G->A)	G to A at 3743	19	Trp to Stop at 1204
W1204X(3744G->A)	G to A at 3744	19	Trp to Stop at 1204
W1274X	G to A at 3954	20	Trp to Stop at 1274
W1282C	G to T at 3978	20	Trp to Cys at 1282
W1282G	T to G at 3976	20	Trp to Gly at 1282
W1282R	T to C at 3976	20	Trp to Arg at 1282
W1282X	G to A at 3978	20	Trp to Stop at 1282
W1310X	G to A at 4061	21	Trp to Stop at 1310
W1316X	G to A at 4079	21	Trp to Stop at 1316
W19C	G to T at 189	2	Trp to Cys at 19
W19X	G to A at 189	2	Trp to Stop at 19
W202X	G to A at 738	6a	Try to Stop at 202
W216C	G to T at 780	6a	Trp to Cys at 216
W216X	G to A at 779	6a	Trp to Stop at 216
W277R	T to A at 961	6b	Trp to Arg at 277
W356S	G to C at 1199	7	Tryptophan to Serine at codon
			356
W356X	G to A at 1200	7	Trp to Stop at 356
W361R(T->A)	T to A at 1213	7	Trp to Arg at 361
W361R(T->C)	T to C at 1213	7	Trp to Arg at 361
W401X(TAG)	G to A at 1334	8	Trp to Stop at 401
W401X(TGA)	G to A at 1335	8	Tip to Stop at 401
W496X	G to A at 1619	10	Trp to Stop at 496
W57G	T to G at 301	3	Trp to Gly at 57
W57R	T to C at 301	3	Trp to Arg at 57
W57X(TAG)	G to A at 302	3	Trp to Stop at 57
W57X(TGA)	G to A at 303	3	Trp to Stop at 57
W679X	G to A at 2168	13	Trp to Stop at 679
W79R	T to C at 367	3	Trp to Arg at 79
W79X	G to A at 368	3	Trp to Stop at 79
W846X	G to A at 2669	14a	Trp to Stop at 846
W846X	G to A at 2670	14a	Trp to Stop at 846
(2670TGG>TGA)
W882X	G to A at 2777	14b	Trp to Stop at 882
Y1014C	A to G at 3173	17a	Tyr to Cys at 1014
Y1032C	A to G at 3227	17a	Tyr to Cys at 1032 (CBAVD)
Y1032N	T to A at 3226	17a	Tyr to Asn at 1032
Y1073C	A to G at 3350	17b	Tyr to Cys at 1073
Y1092C	A to G at 3407	17b	Tyr to Cys at 1092
Y1092H	T to C at 3406	17b	Tyr to His at 1092
Y1092X(C->A)	C to A at 3408	17b	Tyr to Stop at 1092
Y1092X(C->G)	C to G at 3408	17b	Tyr to Stop at 1092
Y109C	A to G at 458	4	Tyr to Cys at 109
Y109N	T to A at 457	4	Tyr to Asn at 109
Y109X	T to A at 459	4	Tyr to Stop at 109
Y1182X	C to G at 3678	19	Tyr to Stop at 1182
Y122C	A to G at 497	4	Tyr to Cys at 122
Y122H	T to C at 496	4	Tyr to His at 122
Y122X	T to A at 498	4	Tyr to Stop at 122
Y1307C	A to G at 4052	21	Tyr to Cys at 1307
Y1307X	T to A at 4053	21	Tyr to Stop at 1307
Y1381H	T to C at 4273	23	Tyr to His at 1381
Y1381X	C to A at 4275	23	Tyr to Stop at 1381
Y161D	T to G at 613	4	Tyr to Asp at 161
Y161N	T to A at 613	4	Tyr to Asn at 161
Y161S	A to C at 614 (together with	4	Tyr to Ser at 161
	612T/A)
Y247X	C to G at 873	6a	Tyr to Stop at 247
Y301C	A to G at 1034	7	Tyr to Cys at 301
Y304X	C to G at 1044	7	Tyr to Stop at 304
Y515H	T to C at 1675	10	Tyr to His at 515
Y517C	A to G at 1682	10	Tyr to Cys at 517
Y563C	A to G at 1820	12	Tyr to Cys at 563
Y563D	T to G at 1819	12	Tyr to Asp at 563
Y563N	T to A at 1819	12	Tyr to Asn at 563
Y569C	A to G at 1838	12	Tyr to Cys at 569
Y569D	T to G at 1837	12	Tyr to Asp at 569
Y569H	T to C at 1837	12	Tyr to His at 569
Y569X	T to A at 1839	12	Tyr to Stop at 569
Y577F	A to T at 1862	12	Tyr to Phe at 577
Y577Y (1863C/T)	C or T at 1863	12	sequence variation (Tyr at 577
			no change)
Y849X	C to A at 2679	14a	Tyr to Stop at 849
Y84H	T to C at 382	3	Tyr to His at 84
Y852X	T to G at 2688	14a	Tyr to stop at 852 (Premature
			termination)
Y89C	A to G at 398	3	Tyr to Cys at 89
Y913C	A to G at 2870	15	Tyr to Cys at 913
Y913X	T to A at 2871	15	Tyr to Stop at 913
Y914C	A to G at 2873	15	Tyr to Cys at 914
Y917C	A to G at 2882	15	Tyr to Cys at 917
Y917D	T to G at 2881	15	Tyr to Asp at 917
Y919C	A to G at 2888	15	Tyr to Cys at 919
*Unless otherwise indicated, the numbers listed herein refer to exon numbers.

TABLE 2
CFTR Mutants and Their Disease Association.
(CF: cystic fibrosis; CBAVD: congenital
bilateral absence of the vas deferens.)

			SWISS-PROT
	Length		Feature Table
Position(s)	(aa)	Description and disease association	Identifier

31	1	R → L in CF. Ref. 44	VAR_000103
42	1	S → F in CF. Ref. 48	VAR_000104
44	1	D → G in CF.	VAR_000105
50	1	S → Y in CBAVD. Ref. 54	VAR_000107
57	1	W → G in CF. Ref. 42	VAR_000108
67	1	P → L in CF.	VAR_000109
74	1	R → W in CF.	VAR_000110
85	1	G → E in CF. Ref. 58	VAR_000112
87	1	F → L in CF. Ref. 39	VAR_000113
91	1	G → R in CF.	VAR_000114
92	1	E → K in CF. Ref. 26 Ref. 29	VAR_000115
98	1	Q → R in CF. Ref. 46	VAR_000116
105	1	I → S in CF.	VAR_000117
109	1	Y → C in CF. Ref. 37	VAR_000118
110	1	D → H in CF.	VAR_000119
111	1	P → L in CBAVD. Ref. 69	VAR_000120
117	1	R → C in CF. Ref. 26 Ref. 48	VAR_000121
		Ref. 58 Ref. 65
117	1	R → H in CF and CBAVD.	VAR_000122
117	1	R → L in CF. Ref. 26 Ref. 48	VAR_000123
		Ref. 58 Ref. 65
117	1	R → P in CF. Ref. 26 Ref. 48	VAR_000124
		Ref. 58 Ref. 65
120	1	A → T in CF. Ref. 38	VAR_000125
139	1	H → R in CF. Ref. 48	VAR_000126
141	1	A → D in CF. Ref. 56	VAR_000127
148	1	I → T in CF. dbSNP rs35516286.	VAR_000128
149	1	G → R in CBAVD. Ref. 40	VAR_000129
178	1	G → R in CF.	VAR_000130
192	1	Missing in CF. Ref. 65	VAR_000131
193	1	E → K in CBAVD and CF.	VAR_000132
199	1	H → Q in CF. Ref. 34	VAR_ 000133
199	1	H → Y in CF. Ref. 34	VAR_000134
205	1	P → S in CF. Ref. 30	VAR_000135
206	1	L → W in CF. Ref. 43	VAR_000136
225	1	C → R in CF.	VAR_000137
244	1	M → K in CBAVD. Ref. 69	VAR_000138
258	1	R → G in CBAVD. Ref. 40	VAR_000139
287	1	N → Y in CF. Ref. 58	VAR_000140
297	1	R → Q in CF.	VAR_000141
301	1	Y → C in CF.	VAR_000142
307	1	S → N in CF.	VAR_000143
311	1	F → L in CF. Ref. 59	VAR_000144
311	1	Missing in CF. Ref. 59	VAR_000145
314	1	G → E in CF. Ref. 50	VAR_000146
314	1	G → R in CF. Ref. 50	VAR_000147
334	1	R → W in CF; mild.	VAR_000148
336	1	I → K in CF.	VAR_000150
338	1	T → I in CF; mild; isolated	VAR_000151
		hypotonic dehydration.
		Ref. 47 Ref. 64
346	1	L → P in CF; dominant	VAR_000152
		mutation but mild phenotype.
		Ref. 33
347	1	R → H in CF.	VAR_000153
347	1	R → L in CF.	VAR_000154
347	1	R → P in CF; MILD.	VAR_000155
352	1	R → Q in CF.	VAR_000156
359	1	Q → K in CF.	VAR_000157
359-360	2	QT → KK in CF.	VAR_000158
370	1	K → KNK in CF.	VAR_000159
455	1	A → E in CF. Ref. 58	VAR_000160
456	1	V → F in CF.	VAR_000161
458	1	G → V in CF.	VAR_000162
480	1	G → C in CF.	VAR_000165
492	1	S → F in CF.	VAR_000166
504	1	E → Q in CF.	VAR_000167
507	1	Missing in CF.	VAR_000170
508	1	Missing in CF and CBAVD;	VAR_000171
		most common mutation;
		72% of the CF patients;
		CFTR fails to be properly
		delivered to plasma membrane.
513	1	D → G in CBAVD. Ref. 68	VAR_000173
520	1	V → F in CF. Ref. 23	VAR_000174
544	1	G → V in CBAVD. Ref. 69	VAR_000175
549	1	S → N in CF.	VAR_000176
549	1	S → I in CF.	VAR_000177
549	1	S → R in CF.	VAR_000178
551	1	G → D in CF. Ref. 58	VAR_000179
551	1	G → S in CF. Ref. 58	VAR_000180
553	1	R → Q in CF.	VAR_000181
558	1	L → S in CF.	VAR_000182
559	1	A → T in CF.	VAR_000183
560	1	R → K in CF. Ref. 63	VAR_000184
560	1	R → S in CF. Ref. 63	VAR_000185
560	1	R → T in CF. Ref. 63	VAR_000186
562	1	V → L in CF. Ref. 53	VAR_000188
563	1	Y → N in CF.	VAR_000189
569	1	Y → C in CF. Ref. 51 Ref. 63	VAR_000190
569	1	Y → D in CF. Ref. 51 Ref. 63	VAR_000191
569	1	Y → H in CF. Ref. 51 Ref. 63	VAR_000192
571	1	L → S in CF.	VAR_000193
572	1	D → N in CF. Ref. 45	VAR_000194
574	1	P → H in CF.	VAR_000195
579	1	D → G in CF. Ref. 42 Ref. 70	VAR_000197
601	1	I → F in CF.	VAR_000198
610	1	L → S in CF.	VAR_000199
613	1	A → T in CF.	VAR_000200
614	1	D → G in CF.	VAR_000201
618	1	I → T in CF.	VAR_000202
619	1	L → S in CF. Ref. 34	VAR_000203
620	1	H → P in CF.	VAR_000204
620	1	H → Q in CF.	VAR_000205
622	1	G → D in oligospermia.	VAR_000206
628	1	G → R in CF.	VAR_000207
633	1	L → P in CF.	VAR_000208
648	1	D → V in CF.	VAR_000209
651	1	D → N in CF.	VAR_000210
665	1	T → S in CF. Ref. 49	VAR_000211
754	1	V → M in CF.	VAR_000214
766	1	R → M in CBAVD.	VAR_000215
792	1	R → G in CBAVD.	VAR_000216
800	1	A → G in CBAVD. Ref. 40	VAR_000217
807	1	I → M in CBAVD.	VAR_000218
		dbSNP rs1800103.
822	1	E → K in CF.	VAR_000219
826	1	E → K in thoracic sarcoidosis.	VAR_000220
866	1	C → Y in CF.	VAR_000221
912	1	S → L Ref. 32	VAR_000222
913	1	Y → C in CF.	VAR_000223
917	1	Y → C in CF.	VAR_000224
949	1	H → Y in CF. Ref. 32	VAR_000225
952	1	M → I in CF.	VAR_000226
997	1	L → F in CF. dbSNP rs1800111.	VAR_000227
1005	1	I → R in CF. Ref. 34	VAR_000228
1006	1	A → E in CF. Ref. 48	VAR_000229
1013	1	P → L in CF. Ref. 60	VAR_000230
1028	1	M → I in CF. Ref. 60	VAR_000231
1052	1	F → V in CF. Ref. 28	VAR_000232
1061	1	G → R in CF. Ref. 28 Ref. 52	VAR_000233
1065	1	L → P in CF. Ref. 32 Ref. 66	VAR_000234
1065	1	L → R in CF. Ref. 32 Ref. 66	VAR_000235
1066	1	R → C in CF. Ref. 28 Ref. 57	VAR_000236
1066	1	R → H in CF. Ref. 28 Ref. 57	VAR_000237
1066	1	R → L in CF. Ref. 28 Ref. 57	VAR_000238
1067	1	A → T in CF.	VAR_000239
1070	1	R → Q in CF. Ref. 28 Ref. 58	VAR_000241
1070	1	R → P in CF. Ref. 28 Ref. 58	VAR_000242
1071	1	Q → P in CF. Ref. 32	VAR_000243
1072	1	P → L in CF.	VAR_000244
1077	1	L → P in CF.	VAR_000245
1085	1	H → R in CF. Ref. 28	VAR_000246
1098	1	W → R in CF. Ref. 44	VAR_000247
1101	1	M → K in CF. Ref. 27 Ref. 28	VAR_000248
1137	1	M → V in CF.	VAR_000249
1140	1	Missing in CF. Ref. 55	VAR_000250
1152	1	D → H in CF.	VAR_000251
1234	1	I → V in CF.	VAR_000254
1235	1	S → R in CF.	VAR_000255
1244	1	G → E in CF.	VAR_000256
1249	1	G → E in CF. Ref. 35	VAR_000257
1251	1	S → N in CF.	VAR_000258
1255	1	S → P in CF. Ref. 25	VAR_000259
1270	1	D → N in CF. dbSNP rs119711167.	VAR_000260
1282	1	W → R in CF.	VAR_000261
1283	1	R → M in CF. Ref. 24	VAR_000262
1286	1	F → S in CF.	VAR_000263
1291	1	Q → H in CF. Ref. 23 Ref. 34	VAR_000264
1291	1	Q → R in CF. Ref. 23 Ref. 34	VAR_000265
1303	1	N → H in CF. Ref. 58	VAR_000266
1303	1	N → K in CF. Ref. 58	VAR_000267
1349	1	G → D in CF.	VAR_000268
1364	1	A → V in CBAVD. Ref. 69	VAR_000269
1397	1	V → E in CF. Ref. 36	VAR_000270
1070	1	R → W in CBAVD.	VAR_011564
1101	1	M → R in CF. Ref. 27 Ref. 28	VAR_011565

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In other aspects, the invention provides methods of making and using the novel cells and cell lines expressing CFTR (e.g., wild type or mutant CFTR). In other aspects, the cells and cell lines of the invention can be used to screen for modulators of CFTR function, including modulators that are specific for a particular form (e.g., mutant form) of CFTR, e.g., modulators that affect CFTR's chloride ion conductance function or CFTR's response to forskolin. These modulators are useful as therapeutics that target, for example, mutant CFTRs in disease states or tissues. CFTR-associated diseases and conditions include, without limitation, cystic fibrosis, lung diseases (e.g., chronic obstructive pulmonary and pulmonary edema), gastrointestinal conditions (e.g., CF pathologies, bowel cleaning, irritable bowel syndrome, constipation, diarrhea, cholera, viral gastroenteritis, malabsorption syndromes, and short bowel syndrome), endocrinal conditions (e.g., pancreatic dysfunction in CF patients), infertility (e.g., sperm motility and sperm capacitation problems and hostile cervical mucus), dry mouth, dry eye, glaucoma, and other deficiencies in regulation of mucosal and/or epithelial fluid absorption and secretion.

In various embodiments, the cell or cell line of the invention expresses CFTR at a consistent level of expression for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 days or over 200 days, where consistent expression refers to a level of expression that does not vary by more than: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% 9% or 10% over 2 to 4 days of continuous cell culture; 2%, 4%, 6%, 8%, 10% or 12% over 5 to 15 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20% over 16 to 20 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24% over 21 to 30 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% over 30 to 40 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% over 41 to 45 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% over 45 to 50 days of continuous cell culture; 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% over 45 to 50 days of continuous cell culture, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30% over 50 to 55 days of continuous cell culture, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30% or 35% over 50 to 55 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% over 55 to 75 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 75 to 100 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 101 to 125 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 126 to 150 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 151 to 175 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over 176 to 200 days of continuous cell culture; 1%, 2%, 3%, 4%, 5%, 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% over more than 200 days of continuous cell culture.

In some embodiments, the cells and cell lines of the invention express a CFTR wherein one or more physiological properties of the cells/cell lines remain(s) substantially constant over time. A physiological property includes any observable, detectable or measurable property of cells or cell lines apart from the expression of the CFTR.

In some embodiments, the expression of CFTR can alter one or more physiological properties. Alteration of a physiological property includes any change of the physiological property due to the expression of CFTR, e.g., a stimulation, activation, or increase of the physiological property, or an inhibition, blocking, or decrease of the physiological property. In these embodiments, the one or more constant physiological properties can indicate that the functional expression of the CFTR also remains constant.

The invention provides a method for culturing a plurality of cells or cell lines expressing a CFTR under constant culture conditions, wherein cells or cell lines can be selected that have one or more desired properties, such as stable expression of a CFTR and/or one or more substantially constant physiological properties.

In some embodiments where a physiological property can be measured, the physiological property is determined as an average of the physiological property measured in a plurality of cells or a plurality of cells of a cell line. In certain embodiments, a physiological property is measured over at least 10; 100; 1,000; 10,000; 100,000; 1,000,000; or at least 10,000,000 cells and the average remains substantially constant over time. In some embodiments, the average of a physiological property is determined by measuring the physiological property in a plurality of cells or a plurality of cells of a cell line wherein the cells are at different stages of the cell cycle. In other embodiments, the cells are synchronized with respect to cell cycle.

In some embodiments, a physiological property is observed, detected, measured or monitored on a single cell level. In certain embodiments, the physiological property remains substantially constant over time on a single cell level.

In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 12 hours. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 1 day. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 2 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 5 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 10 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 20 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 30 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 40 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 50 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 60 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 70 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%; 30%, 35%, 40%, 45%, or 50% over 80 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over 90 days. In certain embodiments, a physiological property remains substantially constant over time if it does not vary by more than 0.1%, 0.5%, 1%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% over the course of 1 passage, 2 passages, 3 passages, 5 passages, 10 passages, 25 passages, 50 passages, or 100 passages.

Examples of cell physiological properties include, but are not limited to: growth rate, size, shape, morphology, volume; profile or content of DNA, RNA, protein, lipid, ion, carbohydrate or water; endogenous, engineered, introduced, gene-activated or total gene, RNA or protein expression or content; propensity or adaptability to growth in adherent, suspension, serum-containing, serum-free, animal-component free, shaken, stationary or bioreactor growth conditions; propensity or adaptability to growth in or on chips, arrays, microarrays, slides, dishes, plates, multiwell plates, high density multiwell plates, flasks, roller bottles, bags or tanks; propensity or adaptability to growth using manual or automated or robotic cell culture methodologies; abundance, level, number, amount or composition of at least one cell organelle, compartment or membrane; including, but not limited to cytoplasm, nucleoli, nucleus, ribosomes, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, centrioles, chloroplasts, cell membrane, plasma cell membrane, nuclear membrane, nuclear envelope, vesicles (e.g., secretory vesicles), or membrane of at least one organelle; having acquired or having the capacity or propensity to acquire at least one functional or gene expression profile (of one or more genes) shared by one or more specific cell types or differentiated, undifferentiated or dedifferentiated cell types, including, but not limited to: a stem cell, a pluripotent cell, an omnipotent cell or a specialized or tissue specific cell including one of the liver, lung, skin, muscle (including but not limited to: cardiac muscle, skeletal muscle, striatal muscle), pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastro-intestinal tract, stomach, thyroid, tongue, gall bladder, kidney, nose, eye, nail, hair, taste bud cell or taste cell, neuron, skin, pancreas, blood, immune, red blood cell, white blood cell, killer T-cell, enteroendocrine cell, secretory cell, kidney, epithelial cell, endothelial cell, a human, animal or plant cell; ability to or capacity to uptake natural or synthetic chemicals or molecules including, but not limited to: nucleic acids, RNA, DNA, protein, small molecules, probes, dyes, oligonucleotides (including modified oligonucleotides) or fluorogenic oligonucleotides; resistance to or capacity to resist negative or deleterious effects of chemicals or substances that negatively affect cell growth, function or viability, including, but not limited to: resistance to infection, drugs, chemicals, pathogens, detergents, UV, adverse conditions, cold, hot, extreme temperatures, shaking, perturbation, vortexing, lack of or low levels of oxygen, lack of or low levels of nutrients, toxins, venoms, viruses or compound, treatment or agent that has an adverse effect on cells or cell growth; suitability for use in in vitro tests, cell based assays, biochemical or biological tests, implantation, cell therapy or secondary assays, including, but not limited to: large scale cell culture, miniaturized cell culture, automated cell culture, robotic cell culture, standardized cell culture, drug discovery, high throughput screening, cell based assay, functional cell based assay (including but not limited to membrane potential assays, calcium flux assays, reporter assays, G-protein reporter assays), ELISA, in vitro assays, in vivo applications, secondary testing, compound testing, binding assays, panning assays, antibody panning assays, phage display, imaging studies; microscopic imaging assays, immunofluorescence studies, RNA, DNA, protein or biologic production or purification, vaccine development, cell therapy, implantation into an organism, animal, human or plant, isolation of factors secreted by the cell, preparation of cDNA libraries, or infection by pathogens, viruses or other agent; and other observable, measurable, or detectable physiological properties such as: biosynthesis of at least one metabolite, lipid, DNA, RNA or protein; chromosomal silencing, activation, heterochromatization, euchromoatinization or recombination; gene expression, gene silencing, gene splicing, gene recombination or gene-activation; RNA production, expression, transcription, processing splicing, transport, localization or modification; protein production, expression, secretion, folding, assembly, transport, localization, cell surface presentation, secretion or integration into a cell or organelle membrane; protein modification including but not limited to post-translational modification, processing, enzymatic modification, proteolysis, glycosylation, phosphorylation, dephosphorylation; cell division including mitosis, meiosis or fission or cell fusion; high level RNA or protein production or yield.

Physiological properties may be observed, detected or measured using routine assays known in the art, including but not limited to tests and methods described in reference guides and manuals such as the Current Protocols series. This series includes common protocols in various fields and is available through the Wiley Publishing House. The protocols in these reference guides are illustrative of the methods that can be used to observe, detect or measure physiological properties of cells. The skilled worker would readily recognize any one or more of these methods may be used to observe, detect or measure the physiological properties disclosed herein.

Many markers, dyes or reporters, including protein markers expressed as fusion proteins comprising an autofluorescent protein, that can be used to measure the level, activity or content of cellular compartments or organelles including but not limited to ribosomes, mitochondria, ER, rER, golgi, TGN, vesicles, endosomes and plasma membranes in cells are compatible with the testing of individual viable cells. In some embodiments fluorescence activated cell sorting or a cell sorter can be used. In some embodiments, cells or cell lines isolated or produced to comprise a CFTR can be tested using these markers, dyes or reporters at the same time, subsequent, or prior to isolation, testing or production of the cells or cell lines comprising a CFTR. In some embodiments, the level, activity or content of one or more of the cellular compartments or organelles can be correlated with improved, increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, yield or physiology of a CFTR. In some embodiments, cells or cell lines comprising the level, activity or content of at least one cellular compartment or organelle that is correlated with improved, increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, yield or physiology of a CFTR can be isolated. In some embodiments, cells or cell lines comprising the CFTR and the level, activity or content of at least one cellular compartment or organelle that is correlated with improved, increased, native, non-cytotoxic, viable or optimal expression, function, activity, folding, assembly modification, post-translational modification, secretion, cell surface presentation, membrane integration, pharmacology, yield or physiology of the CFTR can be isolated. In some embodiments the isolation of the cells is performed using cell sorting or fluorescence activated cell sorting.

The nucleic acid encoding the CFTR can be genomic DNA or cDNA. In some embodiments, the nucleic acid encoding the CFTR comprises one or more substitutions, mutations, or deletions, as compared to a wild type CFTR (SEQ ID NO: 1), that may or may not result in an amino acid substitution. In some embodiments, the nucleic acid is a fragment of the nucleic acid sequence provided. Such CFTR that are fragments or have such modifications retain at least one biological property of a CFTR, e.g., its ability to conduct chloride ions or be modulated by forskolin. The invention encompasses cells and cell lines stably expressing a CFTR-encoding nucleotide sequence that is at least about 85% identical to a sequence disclosed herein. In some embodiments, the CFTR-encoding sequence identity is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher compared to a CFTR sequence provided herein. The invention also encompasses cells and cell lines wherein a nucleic acid encoding a CFTR hybridizes under stringent conditions to a nucleic acid provided herein encoding the CFTR.

In some embodiments, the cell or cell line comprises a CFTR-encoding nucleic acid sequence comprising a substitution compared to a sequence provided herein by at least one but less than 10, 20, 30, or 40 nucleotides, up to or equal to 1%, 5%, 10% or 20% of the nucleotide sequence or a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical thereto, or that is capable of hybridizing under stringent conditions to the sequences disclosed). Such substitutions include single nucleotide polymorphisms (SNPs) and other allelic variations. In some embodiments, the cell or cell line comprises a CFTR-encoding nucleic acid sequence comprising an insertion into or deletion from the sequences provided herein by less than 10, 20, 30, or 40 nucleotides up to or equal to 1%, 5%, 10% or 20% of the nucleotide sequence or from a sequence substantially identical thereto.

In some embodiments, where the nucleic acid substitution or modification results in an amino acid change, such as an amino acid substitution, the native amino acid may be replaced by a conservative or non-conservative substitution (e.g., SEQ ID NO: 7). In some embodiments, the sequence identity between the original and modified polypeptide sequence can differ by about 1%, 5%, 10% or 20% of the polypeptide sequence or from a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identical thereto). Those of skill in the art will understand that a conservative amino acid substitution is one in which the amino acid side chains are similar in structure and/or chemical properties and the substitution should not substantially change the structural characteristics of the parent sequence. In embodiments comprising a nucleic acid comprising a mutation, the mutation may be a random mutation or a site-specific mutation.

Conservative modifications will produce CFTRs having functional and chemical characteristics similar to those of the unmodified CFTR. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties to the parent amino acid residue (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative amino acid substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992). A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

The invention encompasses cells or cell lines that comprise a mutant form of CFTR. More than 1,000 CFTR mutations have been identified, and the cells or cell lines of the invention may comprise any of these mutants of CFTRs. Such cells, cell lines, and collections of cell lines are useful to determine the activity of a mutant CFTR and the differential activity of a modulator on different mutant CFTRs.

The invention further comprises cells or cell lines that co-express other proteins with CFTR. Such other proteins may be integrated into the host cell's genome, or gene-activated, or induced. They may be expressed sequentially (before or after) with respect to CFTR or co-transfected with CFTR on the same or different vectors. In some embodiments, the co-expressed protein may be any of the following: genetic modifiers of CFTR (e.g., al-antitrypsin, glutathione S-transferase, mannose binding lectin 2 (MBL2), nitric oxide synthase 1 (NOS1), glutamine-cysteine ligase gene (GCLC), FCgamma receptor II (FCγRII)); AMP activated protein kinase (AMPK), which phosphorylates and inhibits CFTR and may be important for airway inflammation and ischemia; transforming growth factor β1 (TGF-β1), which downregulates CFTR expression such that coexpression of TGF β1 and CFTR may allow for identifying modulators of this interaction; tumor necrosis factor α (TNF-α), which downregulates CFTR expression such that coexpression TNF-α and CFTR may allow for identifying blockers of this interaction; β adrenergic receptor, which colocalizes with CFTR at the apical membrane and the stimulation of a subtype of β adrenergic receptor (β₂) increases CFTR activity; syntaxin 1a, which inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions and may have therapeutic uses; synaptosome-associated protein 23, which physically associates with and inhibits CFTR; an epithelial sodium ion channel (ENaC), i.e., SCNN1A, SCNN1B or SCNN1G, to study binding interactions that stabilize CFTR at the cell surface; PDZK1 (PDZ domain containing 1) (also referred as CFTR-associated protein of 70 kDa (CAP70)), which potentiates CFTR chloride current; the endocytic complex AP2, which interacts with CFTR and facilitates efficient entry of CFTR into clathrin-coated vesicles; cyclic guanosine monophosphate(cGMP)-dependent protein kinase 2 (PRKG2), which is an upstream cGMP dependent kinase that phosphorylates and activates CFTR; protein kinase A and protein kinase C; protein phosphatase 2 (PP2A); guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 (RACK1); Rho family of GTPases; Rab G TPases, SNARE proteins; potassium channel proteins (e.g., ROMK1 and ROMK2); guanylyl cyclase c (GC-C or GUCY2C), which interacts with CFTR; chloride channel 2 (CLCN2 or CLC2), which is proposed to cause net C1-efflux in gut such that coexpression of both CLCN2 and CFTR may allow for screens demonstrating maximal fluid efflux; solute carrier family 9 isoform A3 (NHE3-SLC9A3/sodium-hydrogen exchanger) or solute carrier family 26 isoform A3 (DRA-SLC26A3/sodium-hydrogen exchanger), to construct a rheostat biosensor for sodium intake/chloride efflux; cyclic nucleotide gated channel (CNGA2), which may be used as a HTS platform with a calcium readout; or a yellow fluorescent protein (YFP or variants thereof such as YFP H148Q/I152L) for usage in YFP halide quench assays.

In some embodiments, the CFTR-encoding nucleic acid sequence further comprises a tag. Such tags may encode, for example, a HIS tag, a myc tag, a hemagglutinin (HA) tag, protein C, VSV-G, FLU, yellow fluorescent protein (YFP), mutant YFP (meYFP), green fluorescent protein (GFP), FLAG, BCCP, maltose binding protein tag, Nus-tag, Softag-1, Softag-2, Strep-tag, S-tag, thioredoxin, GST, V5, TAP or CBP. A tag may be used as a marker to determine CFTR expression levels, intracellular localization, protein-protein interactions, CFTR regulation, or CFTR function. Tags may also be used to purify or fractionate CFTR. One example of a tag is meYFP-H1480/1152L (SEQ ID NO: 5).

Host cells used to produce a cell or cell line of the invention may express endogenous CFTR in its native state or lack expression of any CFTR. The host cell may be a primary, germ, or stem cell, including but not being limited to an embryonic stem cell. The host cell may also be an immortalized cell. Primary or immortalized host cells may be derived from mesoderm, ectoderm or endoderm layers of eukaryotic organisms. The host cell may include but not be limited to endothelial, epidermal, mesenchymal, neural, renal, hepatic, hematopoietic, or immune cells. For example, the host cells may include but not be limited to intestinal crypt or villi cells, clara cells, colon cells, intestinal cells, goblet cells, enterochromafin cells, enteroendocrine cells. The host cells may include but not be limited to be eukaryotic, prokaryotic, mammalian, human, primate, bovine, porcine, feline, rodent, marsupial, murine or other cells. The host cells may also be nonmammalian, including but not being limited to yeast, insect, fungus, plant, lower eukaryotes and prokaryotes. Such host cells may provide backgrounds that are more divergent for testing CFTR modulators with a greater likelihood for the absence of expression products provided by the cell that may interact with the target. In preferred embodiments, the host cell is a mammalian cell. Examples of host cells that may be used to produce a cell or cell line of the invention include but are not limited to: Chinese hamster ovary (CHO) cells, established neuronal cell lines, pheochromocytomas, neuroblastomas fibroblasts, rhabdomyosarcomas, dorsal root ganglion cells, NSO cells, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, HEK-293 (ATCC CRL1573) and PC12 (ATCC CRL-1721), HEK293T (ATCC CRL-11268), RBL (ATCC CRL-1378), SH-SY5Y (ATCC CRL-2266), MDCK (ATCC CCL-34), SJ-RH30 (ATCC CRL-2061), HepG2 (ATCC HB-8065), ND7/23 (ECACC 92090903), CHO (ECACC 85050302), Vero (ATCC CCL 81), Caco-2 (ATCC HTB 37), K562 (ATCC CCL 243), Jurkat (ATCC TIB-152), Per.C6 (Crucell, Leiden, The Netherlands), Huvec (ATCC Human Primary PCS 100-010, Mouse CRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECACC 01042712), 293 (ATCC CRL 10852), A549 (ATCC CCL 185), IMR-90 (ATCC CCL 186), MCF-7 (ATC HTB-22), U-2 OS (ATCC HTB-96), T84 (ATCC CCL 248), or any established cell line (polarized or nonpolarized) or any cell line available from repositories such as the American Type Culture Collection (ATCC, 10801 University Blvd. Manassas, Va. 20110-2209 USA) or European Collection of Cell Cultures (ECACC, Salisbury Wiltshire SP4 0JG England). Host cells used to produce a cell or cell line of the invention may be in suspension. For example, the host cells may be adherent cells adapted to suspension.

In certain embodiments, the methods described herein rely on the genetic variability and diversity in a population of cells, such as a cell line or a culture of immortalized cells. In particular, provided herein are cells, and methods for generating such cells, that express a CFTR endogenously, i.e., without the introduction of a nucleic acid encoding a CFTR. In certain embodiments, the isolated cell expressing the CFTR is represented by not more than 1 in 10, 1 in 100, 1 in 1000, 1 in 10,000, 1 in 100,000, 1 in 1,000,000 or 1 in Ser. No. 10/000,000 cells in a population of cells. The population of cells can be primary cells harvested from organisms. In certain embodiments, the population of cells is not known to express CFTR. In certain embodiments, genetic variability and diversity may also be increased using natural processes known to a person skilled in the art. Any suitable methods for creating or increasing genetic variability and/or diversity may be performed on host cells. In some cases, genetic variability may be due to modifications in regulatory regions of a gene encoding for CFTR. Cells expressing a particular CFTR can then be selected as described herein.

In other embodiments, genetic variability may be achieved by exposing a cell to UV light and/or x-rays (e.g., gamma-rays). In other embodiments, genetic variability may be achieved by exposing cells to EMS (ethyl methane sultonate). In some embodiments, genetic variability may be achieved by exposing cells to mutagens, carcinogens, or chemical agents. Non-limiting examples of such agents include deaminating agents such as nitrous acid, intercalating agents, and alkylating agents. Other non-limiting examples of such agents include bromine, sodium azide, and benzene. In specific embodiments, genetic variability may be achieved by exposing cells to growth conditions that are sub-optimal; e.g., low oxygen, low nutrients, oxidative stress or low nitrogen. In certain embodiments, enzymes that result in DNA damage or that decrease the fidelity of DNA replication or repair (e.g. mismatch repair) can be used to increase genetic variability. In certain embodiments, an inhibitor of an enzyme involved in DNA repair is used. In certain embodiments, a compound that reduces the fidelity of an enzyme involved in DNA replication is used. In certain embodiments, proteins that result in DNA damage and/or decrease the fidelity of DNA replication or repair are introduced into cells (co-expressed, injected, transfected, electroporated).

The duration of exposure to certain conditions or agents depend on the conditions or agents used. In some embodiments, seconds or minutes of exposure is sufficient. In other embodiments, exposure for a period of hours, days or months are necessary. The skilled artisan will be aware what duration and intensity of the condition can be used.

In some cases, a method that increases genetic variability may produce a mutation or alteration in a promoter region of a gene that leads to a change in the transcriptional regulation of the CFTR gene, e.g., gene activation, wherein the gene is more highly expressed than a gene with an unaltered promoter region. Generally, a promoter region includes a genomic DNA sequence upstream of a transcription start site that regulates gene transcription, and may include the minimal promoters and/or enhancers and/or repressor regions. A promoter region may range from about 20 basepairs (bps) to about 10,000 bps or more. In specific embodiments, a method that increases gene variability produces a mutation or alteration in an intron of a CFTR gene that leads to a change in the transcriptional regulation of the gene, e.g., gene activation wherein the gene is more highly expressed than gene with an unaltered intron. In certain embodiments, untranscribed genomic DNA is modified. For example, promoter, enhancer, modifier, or repressor regions can be added, deleted, or modified. In these cases, transcription of a CFTR transcript that is under control of the modified regulatory region can be used as a read-out. For example, if a repressor is deleted, the transcript of the CFTR gene that is repressed by the repressor is tested for increased transcription levels.

In certain embodiments, the genome of a cell or an organism can be mutated by site-specific mutagenesis or homologous recombination. In certain embodiments, oligonucleotide- or triplex-mediated recombination can be employed. See, e.g., Faruqi et al., 2000, Molecular and Cellular Biology 20:990-1000 and Schleifman et al., 2008, Methods Molecular Biology 435:175-90.

In certain embodiments, fluorogenic oligonucleotide probes or molecular beacons can be used to select cells in which the genetic modification has been successful, i.e., cells in which the transgene or the gene of interest is expressed. To identify cells in which a mutagenic or homologous recombination event has been successful, a fluorogenic oligonucleotide that specifically hybridizes to the mutagenized or recombined CFTR transcript can be used.

Once cells that endogenously express CFTR are isolated, these cells can be immortalized and cell lines generated. These cells or cell lines can be used with the assays and screening methods disclosed herein.

In one embodiment, the host cell is an embryonic stem cell that is then used as the basis for the generation of transgenic animals. Embryonic stem cells stably expressing CFTR, and preferably a functional introduced CFTR, may be implanted into organisms directly, or their nuclei may be transferred into other recipient cells and these may then be implanted, or they may be used to create transgenic animals.

As will be appreciated by those of skill in the art, any vector that is suitable for use with the host cell may be used to introduce a nucleic acid encoding CFTR into the host cell. Examples of vectors that may be used to introduce the CFTR encoding nucleic acids into host cells include but are not limited to plasmids, viruses, including retroviruses and lentiviruses, cosmids, artificial chromosomes and may include for example, pFN11A (BIND) Flexi®, pGL4.31, pFC14A (HaloTag® 7) CMV Flexi®, pFC14K (HaloTag® 7) CMV Flexi®, pFN24A (HaloTag® 7) CMVd3 Flexi®, pFN24K (HaloTag® 7) CMVd3 Flexi®, HaloTag™ pHT2, pACT, pAdVAntage™, pALTER®-MAX, pBIND, pCAT®3-Basic, pCAT®3-Control, pCAT®3-Enhancer, pCAT®3-Promoter, pCI,pCMVTNT™, pG5luc, pSI, pTARGET™, pTNT™, pF12A RM Flexi®, pF12K RM Flexi®, pReg neo, pYES2/GS, pAd/CMV/V5-DEST Gateway® Vector, pAd/PL-DEST™ Gateway® Vector, Gateway® pDEST™27 Vector,Gateway® pEF-DEST51 Vector, Gateway® pcDNA™-DEST47 vector, pCMV/Bsd Vector, pEF6/His A, B, & C, pcDNA™6.2-DEST, pLenti6/TR, pLP-AcGFP1-C, pLPS-AcGFP1-N, pLP-IRESneo, pLP-TRE2, pLP-RevTRE, pLP-LNCX, pLP-CMV-HA, pLP-CMV-Myc, pLP-RetroQ, pLP-CMVneo, pCMV-Script, pcDNA3.1 Hygro, pcDNA3.1neo, pcDNA3.1puro, pSV2neo, pIRES puro, and pSV2 zeo. In some embodiments, the vectors comprise expression control sequences such as constitutive or conditional promoters. One of ordinary skill in the art will be able to select such sequences. For example, suitable promoters include but are not limited to CMV, TK, SV40, and EF-1α. In some embodiments, the promoters are inducible, temperature regulated, tissue specific, repressible, heat-shock, developmental, cell lineage specific, eukaryotic, prokaryotic or temporal promoters or a combination or recombination of unmodified or mutagenized, randomized, shuffled sequences of any one or more of the above. In other embodiments, CFTR is expressed by gene activation or when a gene encoding a CFTR is episomal. Nucleic acids encoding CFTRs may preferably be constitutively expressed.

In some embodiments, the vector encoding CFTR lacks a selectable marker or drug resistance gene. In other embodiments, the vector optionally comprises a nucleic acid encoding a selectable marker such as a protein that confers drug or antibiotic resistance. If more than one of the drug resistance markers are the same, simultaneous selection may be achieved by increasing the level of the drug. Suitable markers will be well-known to those of skill in the art and include but are not limited to genes conferring resistance to any one of the following: Neomycin/G418, Puromycin, hygromycin, Zeocin, methotrexate and blasticidin. Although drug selection (or selection using any other suitable selection marker) is not a required step, it may be used to enrich the transfected cell population for stably transfected cells, provided that the transfected constructs are designed to confer drug resistance. If subsequent selection of cells expressing CFTR is accomplished using signaling probes, selection too soon following transfection can result in some positive cells that may only be transiently and not stably transfected. However, this can be minimized by allowing sufficient cell passage allowing for dilution of transient expression in transfected cells.

In some embodiments, the vector comprises a nucleic acid sequence encoding an RNA tag sequence. “Tag sequence” refers to a nucleic acid sequence that is an expressed RNA or portion of an RNA that is to be detected by a signaling probe. Signaling probes may detect a variety of RNA sequences. Any of these RNAs may be used as tags. Signaling probes may be directed against the RNA tag by designing the probes to include a portion that is complementary to the sequence of the tag. The tag sequence may be a 3′ untranslated region of the plasmid that is cotranscribed and comprises a target sequence for signaling probe binding. The RNA encoding the gene of interest may include the tag sequence or the tag sequence may be located within a 5′-untranslated region or 3′-untranslated region. In some embodiments, the tag is not with the RNA encoding the gene of interest. The tag sequence can be in frame with the protein-coding portion of the message of the gene or out of frame with it, depending on whether one wishes to tag the protein produced. Thus, the tag sequence does not have to be translated for detection by the signaling probe. The tag sequences may comprise multiple target sequences that are the same or different, wherein one signaling probe hybridizes to each target sequence. The tag sequences may encode an RNA having secondary structure. The structure may be a three-arm junction structure. Examples of tag sequences that may be used in the invention, and to which signaling probes may be prepared, include but are not limited to the RNA transcript of epitope tags such as, for example, a HIS tag, a myc tag, a hemagglutinin (HA) tag, protein C, VSV-G, FLU, yellow fluorescent protein (YFP), green fluorescent protein, FLAG, BCCP, maltose binding protein tag, Nus-tag, Softag-1, Softag-2, Strep-tag, S-tag, thioredoxin, GST, V5, TAP or CBP. As described herein, one of ordinary skill in the art could create his or her own RNA tag sequences.

In another aspect of the invention, cells and cell lines of the invention have enhanced stability as compared to cells and cell lines produced by conventional methods. To identify stable expression, a cell or cell line's expression of CFTR is measured over a time course and the expression levels are compared. Stable cell lines will continue expressing CFTR throughout the time course. In some aspects of the invention, the time course may be for at least one week, two weeks, three weeks, etc., or at least one month, or at least two, three, four, five, six, seven, eight or nine months, or any length of time in between. Isolated cells and cell lines can be further characterized, such as by qRT-PCR and single end-point RT-PCR to determine the absolute amounts and relative amounts of CFTR being expressed. In some embodiments, stable expression is measured by comparing the results of functional assays over a time course. The measurement of stability based on functional assay provides the benefit of identifying clones that not only stably express the mRNA of the gene of interest, but also stably produce and properly process (e.g., post-translational modification, and localization within the cell) the protein encoded by the gene of interest that functions appropriately.

Cells and cell lines of the invention have the further advantageous property of providing assays with high reproducibility as evidenced by their Z′ factor. See Zhang J H, Chung T D, Oldenburg K R, “A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.” J. Biomol. Screen. 1999; 4(2):67-73. Z′ values pertain to the quality of a cell or cell line because it reflects the degree to which a cell or cell line will respond consistently to modulators. Z′ is a statistical calculation that takes into account the signal-to-noise range and signal variability (i.e., from well to well) of the functional response to a reference compound across a multiwell plate. Z′ is calculated using data obtained from multiple wells with a positive control and multiple wells with a negative control. The ratio of their summated standard deviations multiplied by a factor of three to the difference in their mean values is subtracted from one to give the Z′ factor, according the equation below:

Z′ factor=1−3σ_{positive control}+3σ_{negative control})/(μ_{positive control}−μ_{negative control}))

The theoretical maximum Z′ factor is 1.0, which would indicate an ideal assay with no variability and limitless dynamic range. As used herein, a “high Z′” refers to a Z′ factor of Z′ of at least 0.6, at least 0.7, at least 0.75 or at least 0.8, or any decimal in between 0.6 and 1.0. A score less than 0 is undesirable because it indicates that there is overlap between positive and negative controls. In the industry, for simple cell-based assays, Z′ scores up to 0.3 are considered marginal scores, Z′ scores between 0.3 and 0.5 are considered acceptable, and Z′ scores above 0.5 are considered excellent. Cell-free or biochemical assays may approach higher Z′ scores, but Z′ factor scores for cell-based systems tend to be lower because cell-based systems are complex.

As those of ordinary skill in the art will recognize, historically, cell-based assays using cells expressing even a single chain protein do not typically achieve a Z′ higher than 0.5 to 0.6. Cells and cell lines of the invention, on the other hand, have high Z′ values and advantageously produce consistent results in assays. CFTR expression cells and cell lines of the invention provided the basis for high-throughput screening (HTS) compatible assays because they generally have Z′ factor factors at least 0.82. In some aspects of the invention, the cells and cell lines result in Z′ of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, or at least 0.8. In other aspects of the invention, the cells and cell lines of the invention result in a Z′ of at least 0.7, at least 0.75 or at least 0.8 maintained for multiple passages, e.g., between 5-20 passages, including any integer in between 5 and 20. In some aspects of the invention, the cells and cell lines result in a Z′ of at least 0.7, at least 0.75 or at least 0.8 maintained for 1, 2, 3, 4 or 5 weeks or 2, 3, 4, 5, 6, 7, 8 or 9 months, including any period of time in between.

Also according to the invention, cells and cell lines that express a form of a naturally occurring wild type CFTR or mutant CFTR can be characterized for chloride ion conductance. In some embodiments, the cells and cell lines of the invention express CFTR with “physiologically relevant” activity. As used herein, physiological relevance refers to a property of a cell or cell line expressing a CFTR whereby the CFTR conducts chloride ions as a naturally occurring CFTR of the same type and responds to modulators in the same ways that naturally occurring CFTR of the same type is modulated by the same modulators. CFTR-expressing cells and cell lines of this invention preferably demonstrate comparable function to cells that normally express native CFTR in a suitable assay, such as a membrane potential assay or a YFP halide quench assay using chloride or iodide as the ion conducted by CFTR, electrophysiology (e.g., patch clamp or Ussing), or by activation with forskolin. Such comparisons are used to determine a cell or cell line's physiological relevance.

In some embodiments, the cells and cell lines of the invention have increased sensitivity to modulators of CFTR. Cells and cell lines of the invention respond to modulators and conduct chloride ions with physiological range EC₅₀ or IC₅₀ values for CFTR. As used herein, EC₅₀ refers to the concentration of a compound or substance required to induce a half-maximal activating response in the cell or cell line. As used herein, IC₅₀ refers to the concentration of a compound or substance required to induce a half-maximal inhibitory response in the cell or cell line. EC₅₀ and IC₅₀ values may be determined using techniques that are well-known in the art, for example, a dose-response curve that correlates the concentration of a compound or substance to the response of the CFTR-expressing cell line. For example, the EC₅₀ for forskolin in a cell line of the invention is about 250 nM, and the EC₅₀ for forskolin in a stable CFTR-expressing fisher rat thyroid cell line disclosed in Galietta et al., Am J Physiol Cell Physiol. 281(5): C1734-1742 (2001).is between 250 nM and 500 nM.

A further advantageous property of the CFTR-expressing cells and cell lines of the inventions, flowing from the physiologically relevant function of the CFTR is that modulators identified in initial screening are functional in secondary functional assays, e.g., membrane potential assay, electrophysiology assay, YFP halide quench assay, radioactive iodine flux assay, rabbit intestinal-loop fluid secretion measurement assay, animal fecal output testing and measuring assay, or Ussing chamber assays. As those of ordinary skill in the art will recognize, compounds identified in initial screening assays typically must be modified, such as by combinatorial chemistry, medicinal chemistry or synthetic chemistry, for their derivatives or analogs to be functional in secondary functional assays. However, due to the high physiological relevance of the present CFTR cells and cell lines, many compounds identified therewith are functional without “coarse” tuning.

In some embodiments, properties of the cells and cell lines of the invention, such as stability, physiological relevance, reproducibility in an assay (Z′), or physiological EC₅₀ or IC₅₀ values, are achievable under specific culture conditions. In some embodiments, the culture conditions are standardized and rigorously maintained without variation, for example, by automation. Culture conditions may include any suitable conditions under which the cells or cell lines are grown and may include those known in the art. A variety of culture conditions may result in advantageous biological properties for any of the bitter receptors, or their mutants or allelic variants.

In other embodiments, the cells and cell lines of the invention with desired properties, such as stability, physiological relevance, reproducibility in an assay (Z′), or physiological EC₅₀ or IC₅₀ values, can be obtained within one month or less. For example, the cells or cell lines may be obtained within 2, 3, 4, 5, or 6 days, or within 1, 2, 3 or 4 weeks, or any length of time in between.

One aspect of the invention provides a collection or panel of cells and cell lines, each expressing a different form of CFTR (e.g., wild type, allelic variants, mutants, fragment, spliced variants etc.). The collection may include, for example, cells or cell lines expressing CFTR, CFTR ΔF508 and various other known mutant CFTRs. In some embodiment, the collections or panels include cells expressing other ion channel proteins. The collections or panels may additional comprise cells expressing control proteins. The collections or panels of the invention can be used for compound screening or profiling, e.g., to identify modulators that are active on some or all.

When collections or panels of cells or cell lines are produced, e.g., for drug screening, the cells or cell lines in the collection or panel may be derived from the same host cells and may further be matched such that they are the same (including substantially the same) with regard to one or more selective physiological properties. The “same physiological property” in this context means that the selected physiological property is similar enough amongst the members in the collection or panel such that the cell collection or panel can produce reliable results in drug screening assays; for example, variations in readouts in a drug screening assay will be due to, e.g., the different biological activities of test compounds on cells expressing different forms of CFTR, rather than due to inherent variations in the cells. For example, the cells or cell lines may be matched to have the same growth rate, i.e., growth rates with no more than one, two, three, four, or five hours difference amongst the members of the cell collection or panel. This may be achieved by, for example, binning cells by their growth rate into five, six, seven, eight, nine, or ten groups, and creating a panel using cells from the same binned group. Methods of determining cell growth rate are well known in the art. The cells or cell lines in a panel also can be matched to have the same Z′ factor (e.g., Z′ factors that do not differ by more than 0.1), CFTR expression level (e.g., CFTR expression levels that do not differ by more than 5%, 10%, 15%, 20%, 25%, or 30%), adherence to tissue culture surfaces, and the like. Matched cells and cell lines can be grown under identical conditions, achieved by, e.g., automated parallel processing, to maintain the selected physiological property.

Matched cell panels of the invention can be used to, for example, identify modulators with defined activity (e.g., agonist or antagonist) on CFTR; to profile compound activity across different forms of CFTR; to identify modulators active on just one form of CFTR; and to identify modulators active on just a subset of CFTRs. The matched cell panels of the invention allow high throughput screening. Screenings that used to take months to accomplish can now be accomplished within weeks.

To make cells and cell lines of the invention, one can use, for example, the technology described in U.S. Pat. No. 6,692,965 and International Patent Publication WO/2005/079462. Both of these documents are incorporated herein by reference in their entirety for all purposes. This technology provides real-time assessment of millions of cells such that any desired number of clones (from hundreds to thousands of clones) may be selected. Using cell sorting techniques, such as flow cytometric cell sorting (e.g., with a FACS machine), magnetic cell sorting (e.g., with a MACS machine), or other fluorescence plate readers, including those that are compatible with high-throughput screening, one cell per well may be automatically deposited with high statistical confidence in a culture vessel (such as a 96 well culture plate). The speed and automation of the technology allows multigene cell lines to be readily isolated.

In some embodiments, the invention provides a panel of cell lines comprising at least 3, 5, 10, 25, 50, 100, 250, 500, 750, or 1000 cells or cell lines, each expressing a different CFTR mutant selected from the CFTR mutants set forth in Table 1 or Table 2. In certain embodiments, such a panel comprises at least 3, 5, 10, 25, 50, or 75 cells or cell lines, each expressing a different CFTR mutant selected from the CFTR mutants set forth in Table 2. For example, the panel may comprise a CFTR-ΔF508 expressing cell line. In certain embodiments, the panel comprises at least 3, 5, 10, 25, 50, or 100 cells or cell lines, each expressing a different CFTR mutant, wherein each CFTR mutant is a missense, nonsense, frameshift or RNA splicing mutation. In certain embodiments, such a panel comprises at least 3, 5, 10, 25, 50, or 100 cells or cell lines, each expressing a different CFTR mutant, wherein each CFTR mutant is associated with cystic fibrosis. In certain embodiments, such a panel comprises at least 3, 5, 10, 25, 50, or 100 cells or cell lines each expressing a different CFTR mutant, wherein each CFTR mutant is associated with congenital bilateral absence of the vas deferens. Such panels can be used for parallel high-throughput screening and cross-comparative characterization of small molecules with efficacy against the various isoforms of the CFTR protein. In certain embodiments, such a panel also comprises one or more cells or cell lines engineered or selected to express a protein of interest other than CFTR or CFTR mutant.

Using the technology, the RNA sequence for CFTR may be detected using a signaling probe, also referred to as a molecular beacon or fluorogenic probe. As described in, e.g., U.S. Pat. No. 6,692,965, a molecular beacon typically is a nucleic acid probe that recognizes and reports the presence of a specific nucleic acid sequence. The probes may be hairpin-shaped sequences with a central stretch of nucleotides complementary to the target sequence, and termini comprising short mutually complementary sequences. One terminus is covalently bound to a fluorophore and the other to a quenching moiety. When in their native state with hybridized termini, the proximity of the fluorophore and the quencher is such that no fluorescence is produced. The beacon undergoes a spontaneous fluorogenic conformational change when hybridized to its target nucleic acid. In some embodiments, the molecular beacon (or fluorogenic probe) recognizes a target tag sequence as described above. In another embodiment, the molecular beacon (or fluorogenic probe) recognizes a sequence within CFTR itself. Signaling probes may be directed against the RNA tag or CFTR sequence by designing the probes to include a portion that is complementary to the RNA sequence of the tag or the CFTR, respectively.

Nucleic acids comprising a sequence encoding a CFTR, or the sequence of a CFTR and a tag sequence, and optionally a nucleic acid encoding a selectable marker may be introduced into selected host cells by well known methods. The methods include but are not limited to transfection, viral delivery, protein or peptide mediated insertion, coprecipitation methods, lipid based delivery reagents (lipofection), cytofection, lipopolyamine delivery, dendrimer delivery reagents, electroporation or mechanical delivery. Examples of transfection reagents are GENEPORTER, GENEPORTER2, LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, FUGENE® 6, FUGENE® HD, TFX™-10, TFX™-20, TFX™-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROJENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNAI SHUTTLE, METAFECTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, JETSI, JETPEI, MEGAFECTIN, POLYFECT, TRANSMESSANGER, RNAiFECT, SUPERFECT, EFFECTENE, TF-PEI-KIT, CLONFECTIN, AND METAFECTINE.

Following introduction of the CFTR coding sequences or the CFTR activation sequences into host cells and optional subsequent drug selection, molecular beacons (e.g., fluorogenic probes) are introduced into the cells and cell sorting is used to isolate cells positive for their signals. Multiple rounds of sorting may be carried out, if desired. In one embodiment, the flow cytometric cell sorter is a FACS machine. MACS (magnetic cell sorting) or laser ablation of negative cells using laser-enabled analysis and processing can also be used. Other fluorescence plate readers, including those that are compatible with high-throughput screening can also be used. According to this method, cells expressing CFTR are detected and recovered. The CFTR sequence may be integrated at different locations of the genome in the cell. The expression level of the introduced genes encoding the CFTR may vary based upon integration site. The skilled worker will recognize that sorting can be gated for any desired expression level. Further, stable cell lines may be obtained wherein one or more of the introduced genes encoding a CFTR is episomal or results from gene activation.

Signaling probes useful in this invention are known in the art and generally are oligonucleotides comprising a sequence complementary to a target sequence and a signal emitting system so arranged that no signal is emitted when the probe is not bound to the target sequence and a signal is emitted when the probe binds to the target sequence. By way of non-limiting illustration, the signaling probe may comprise a fluorophore and a quencher positioned in the probe so that the quencher and fluorophore are brought together in the unbound probe. Upon binding between the probe and the target sequence, the quencher and fluorophore separate, resulting in emission of signal. International publication WO/2005/079462, for example, describes a number of signaling probes that may be used in the production of the cells and cell lines of this invention.

Nucleic acids encoding signaling probes may be introduced into the selected host cell by any of numerous means that will be well-known to those of skill in the art, including but not limited to transfection, coprecipitation methods, lipid based delivery reagents (lipofection), cytofection, lipopolyamine delivery, dendrimer delivery reagents, electroporation or mechanical delivery. Examples of transfection reagents are GENEPORTER, GENEPORTER2, LIPOFECTAMINE, LIPOFECTAMINE 2000, FUGENE 6, FUGENE HD, TFX-10, TFX-20, TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROJENE, GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR, SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNAI SHUTTLE, METAFECTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE, JETSI, JETPEI, MEGAFECTIN, POLYFECT, TRANSMESSANGER, RNAiFECT, SUPERFECT, EFFECTENE, TF-PEI-KIT, CLONFECTIN, AND METAFECTINE.

In one embodiment, the signaling probes are designed to be complementary to either a portion of the RNA encoding a CFTR or to portions of their 5′ or 3′ untranslated regions. Even if the signaling probe designed to recognize a messenger RNA of interest is able to detect spuriously endogenously existing target sequences, the proportion of these in comparison to the proportion of the sequence of interest produced by transfected cells is such that the sorter is able to discriminate the two cell types.

The expression level of CFTR may vary from cell or cell line to cell or cell line. The expression level in a cell or cell line also may decrease over time due to epigenetic events such as DNA methylation and gene silencing and loss of transgene copies. These variations can be attributed to a variety of factors, for example, the copy number of the transgene taken up by the cell, the site of genomic integration of the transgene, and the integrity of the transgene following genomic integration. One may use FACS or other cell sorting methods (i.e., MACS) to evaluate expression levels. Additional rounds of introducing signaling probes may be used, for example, to determine if and to what extent the cells remain positive over time for any one or more of the RNAs for which they were originally isolated.

In another embodiment of the invention, adherent cells can be adapted to suspension before or after cell sorting and isolating single cells. In other embodiments, isolated cells may be grown individually or pooled to give rise to populations of cells. Individual or multiple cell lines may also be grown separately or pooled. If a pool of cell lines is producing a desired activity or has a desired property, it can be further fractionated until the cell line or set of cell lines having this effect is identified. Pooling cells or cell lines may make it easier to maintain large numbers of cell lines without the requirements for maintaining each separately. Thus, a pool of cells or cell lines may be enriched for positive cells. An enriched pool may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% are positive for the desired property or activity.

In a further aspect, the invention provides a method for producing the cells and cell lines of the invention. In one embodiment, the method comprises the steps of:

• • a) providing a plurality of cells that express mRNA encoding CFTR;
  • b) dispersing cells individually into individual culture vessels, thereby providing a plurality of separate cell cultures
  • c) culturing the cells under a set of desired culture conditions using automated cell culture methods characterized in that the conditions are substantially identical for each of the separate cell cultures, during which culturing the number of cells in each separate cell culture is normalized, and wherein the separate cultures are passaged on the same schedule;

d) assaying the separate cell cultures for at least one desired characteristic of CFTR at least twice; and

• • e) identifying a separate cell culture that has the desired characteristic in both assays.

According to the method, the cells are cultured under a desired set of culture conditions. The conditions can be any desired conditions. Those of skill in the art will understand what parameters are comprised within a set of culture conditions. For example, culture conditions include but are not limited to: the media (Base media (DMEM, MEM, RPMI, serum-free, with serum, fully chemically defined, without animal-derived components), mono and divalent ion (sodium, potassium, calcium, magnesium) concentration, additional components added (amino acids, antibiotics, glutamine, glucose or other carbon source, HEPES, channel blockers, modulators of other targets, vitamins, trace elements, heavy metals, co-factors, growth factors, anti-apoptosis reagents), fresh or conditioned media, with HEPES, pH, depleted of certain nutrients or limiting (amino acid, carbon source)), level of confluency at which cells are allowed to attain before split/passage, feeder layers of cells, or gamma-irradiated cells, CO₂, a three gas system (oxygen, nitrogen, carbon dioxide), humidity, temperature, still or on a shaker, and the like, which will be well known to those of skill in the art.

The cell culture conditions may be chosen for convenience or for a particular desired use of the cells. Advantageously, the invention provides cells and cell lines that are optimally suited for a particular desired use. That is, in embodiments of the invention in which cells are cultured under conditions for a particular desired use, cells are selected that have desired characteristics under the condition for the desired use.

By way of illustration, if cells will be used in assays in plates where it is desired that the cells are adherent, cells that display adherence under the conditions of the assay may be selected. Similarly, if the cells will be used for protein production, cells may be cultured under conditions appropriate for protein production and selected for advantageous properties for this use.

In some embodiments, the method comprises the additional step of measuring the growth rates of the separate cell cultures. Growth rates may be determined using any of a variety of techniques means that will be well known to the skilled worker. Such techniques include but are not limited to measuring ATP, cell confluency, light scattering, optical density (e.g., OD 260 for DNA). Preferably growth rates are determined using means that minimize the amount of time that the cultures spend outside the selected culture conditions.

In some embodiments, cell confluency is measured and growth rates are calculated from the confluency values. In some embodiments, cells are dispersed and clumps removed prior to measuring cell confluency for improved accuracy. Means for monodispersing cells are well-known and can be achieved, for example, by addition of a dispersing reagent to a culture to be measured. Dispersing agents are well-known and readily available, and include but are not limited to enzymatic dispering agents, such as trypsin, and EDTA-based dispersing agents. Growth rates can be calculated from confluency date using commercially available software for that purpose such as HAMILTON VECTOR. Automated confluency measurement, such as using an automated microscopic plate reader is particularly useful. Plate readers that measure confluency are commercially available and include but are not limited to the CLONE SELECT IMAGER (Genetix). Typically, at least 2 measurements of cell confluency are made before calculating a growth rate. The number of confluency values used to determine growth rate can be any number that is convenient or suitable for the culture. For example, confluency can be measured multiple times over e.g., a week, 2 weeks, 3 weeks or any length of time and at any frequency desired.

When the growth rates are known, according to the method, the plurality of separate cell cultures are divided into groups by similarity of growth rates. By grouping cultures into growth rate bins, one can manipulate the cultures in the group together, thereby providing another level of standardization that reduces variation between cultures. For example, the cultures in a bin can be passaged at the same time, treated with a desired reagent at the same time, etc. Further, functional assay results are typically dependent on cell density in an assay well. A true comparison of individual clones is only accomplished by having them plated and assayed at the same density. Grouping into specific growth rate cohorts enables the plating of clones at a specific density that allows them to be functionally characterized in a high throughput format.

The range of growth rates in each group can be any convenient range. It is particularly advantageous to select a range of growth rates that permits the cells to be passaged at the same time and avoid frequent renormalization of cell numbers. Growth rate groups can include a very narrow range for a tight grouping, for example, average doubling times within an hour of each other. But according to the method, the range can be up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours or up to 10 hours of each other or even broader ranges. The need for renormalization arises when the growth rates in a bin are not the same so that the number of cells in some cultures increases faster than others. To maintain substantially identical conditions for all cultures in a bin, it is necessary to periodically remove cells to renormalize the numbers across the bin. The more disparate the growth rates, the more frequently renormalization is needed.

In step d) the cells and cell lines may be tested for and selected for any physiological property including but not limited to: a change in a cellular process encoded by the genome; a change in a cellular process regulated by the genome; a change in a pattern of chromosomal activity; a change in a pattern of chromosomal silencing; a change in a pattern of gene silencing; a change in a pattern or in the efficiency of gene activation; a change in a pattern or in the efficiency of gene expression; a change in a pattern or in the efficiency of RNA expression; a change in a pattern or in the efficiency of RNAi expression; a change in a pattern or in the efficiency of RNA processing; a change in a pattern or in the efficiency of RNA transport; a change in a pattern or in the efficiency of protein translation; a change in a pattern or in the efficiency of protein folding; a change in a pattern or in the efficiency of protein assembly; a change in a pattern or in the efficiency of protein modification; a change in a pattern or in the efficiency of protein transport; a change in a pattern or in the efficiency of transporting a membrane protein to a cell surface change in growth rate; a change in cell size; a change in cell shape; a change in cell morphology; a change in % RNA content; a change in % protein content; a change in % water content; a change in % lipid content; a change in ribosome content; a change in mitochondrial content; a change in ER mass; a change in plasma membrane surface area; a change in cell volume; a change in lipid composition of plasma membrane; a change in lipid composition of nuclear envelope; a change in protein composition of plasma membrane; a change in protein; composition of nuclear envelope; a change in number of secretory vesicles; a change in number of lysosomes; a change in number of vacuoles; a change in the capacity or potential of a cell for: protein production, protein secretion, protein folding, protein assembly, protein modification, enzymatic modification of protein, protein glycosylation, protein phosphorylation, protein dephosphorylation, metabolite biosynthesis, lipid biosynthesis, DNA synthesis, RNA synthesis, protein synthesis, nutrient absorption, cell growth, mitosis, meiosis, cell division, to dedifferentiate, to transform into a stem cell, to transform into a pluripotent cell, to transform into a omnipotent cell, to transform into a stem cell type of any organ (i.e., liver, lung, skin, muscle, pancreas, brain, testis, ovary, blood, immune system, nervous system, bone, cardiovascular system, central nervous system, gastro-intestinal tract, stomach, thyroid, tongue, gall bladder, kidney, nose, eye, nail, hair, taste bud), to transform into a differentiated any cell type (i.e., muscle, heart muscle, neuron, skin, pancreatic, blood, immune, red blood cell, white blood cell, killer T-cell, enteroendocrine cell, taste, secretory cell, kidney, epithelial cell, endothelial cell, also including any of the animal or human cell types already listed that can be used for introduction of nucleic acid sequences), to uptake DNA, to uptake small molecules, to uptake fluorogenic probes, to uptake RNA, to adhere to solid surface, to adapt to serum-free conditions, to adapt to serum-free suspension conditions, to adapt to scaled-up cell culture, for use for large scale cell culture, for use in drug discovery, for use in high throughput screening, for use in a functional cell based assay, for use in membrane potential assays, for use in reporter cell based assays, for use in ELISA studies, for use in in vitro assays, for use in vivo applications, for use in secondary testing, for use in compound testing, for use in a binding assay, for use in panning assay, for use in an antibody panning assay, for use in imaging assays, for use in microscopic imaging assays, for use in multiwell plates, for adaptation to automated cell culture, for adaptation to miniaturized automated cell culture, for adaptation to large-scale automated cell culture, for adaptation to cell culture in multiwell plates (6, 12, 24, 48, 96, 384, 1536 or higher density), for use in cell chips, for use on slides, for use on glass slides, for microarray on slides or glass slides, for immunofluorescence studies, for use in protein purification, and for use in biologics production. Those of skill in the art will readily recognize suitable tests for any of the above-listed properties.

Tests that may be used to characterize cells and cell lines of the invention and/or matched panels of the invention include but are not limited to: amino acid analysis, DNA sequencing, protein sequencing, NMR, a test for protein transport, a test for nucelocytoplasmic transport, a test for subcellular localization of proteins, a test for subcellular localization of nucleic acids, microscopic analysis, submicroscopic analysis, fluorescence microscopy, electron microscopy, confocal microscopy, laser ablation technology, cell counting and Dialysis. The skilled worker would understand how to use any of the above-listed tests.

According to the method, cells may be cultured in any cell culture format so long as the cells or cell lines are dispersed in individual cultures prior to the step of measuring growth rates. For example, for convenience, cells may be initially pooled for culture under the desired conditions and then individual cells separated one cell per well or vessel. Cells may be cultured in multi-well tissue culture plates with any convenient number of wells. Such plates are readily commercially available and will be well knows to a person of skill in the art. In some cases, cells may preferably be cultured in vials or in any other convenient format, the various formats will be known to the skilled worker and are readily commercially available.

In embodiments comprising the step of measuring growth rate, prior to measuring growth rates, the cells are cultured for a sufficient length of time for them to acclimate to the culture conditions. As will be appreciated by the skilled worker, the length of time will vary depending on a number of factors such as the cell type, the chosen conditions, the culture format and may be any amount of time from one day to a few days, a week or more.

Preferably, each individual culture in the plurality of separate cell cultures is maintained under substantially identical conditions a discussed below, including a standardized maintenance schedule. Another advantageous feature of the method is that large numbers of individual cultures can be maintained simultaneously, so that a cell with a desired set of traits may be identified even if extremely rare. For those and other reasons, according to the invention, the plurality of separate cell cultures are cultured using automated cell culture methods so that the conditions are substantially identical for each well. Automated cell culture prevents the unavoidable variability inherent to manual cell culture.

Any automated cell culture system may be used in the method of the invention. A number of automated cell culture systems are commercially available and will be well-known to the skilled worker. In some embodiments, the automated system is a robotic system. Preferably, the system includes independently moving channels, a multichannel head (e.g., a 96-tip head) and a gripper or cherry-picking arm and a HEPA filtration device to maintain sterility during the procedure. The number of channels in the pipettor should be suitable for the format of the culture. Convenient pipettors have, e.g., 96 or 384 channels. Such systems are known and are commercially available. For example, a MICROLAB START™ instrument (Hamilton) may be used in the method of the invention. The automated system should be able to perform a variety of desired cell culture tasks. Such tasks will be known by a person of skill in the art. They include but are not limited to: removing media, replacing media, adding reagents, cell washing, removing wash solution, adding a dispersing agent, removing cells from a culture vessel, adding cells to a culture vessel an the like.

The production of a cell or cell line of the invention may include any number of separate cell cultures. However, the advantages provided by the method increase as the number of cells increases. There is no theoretical upper limit to the number of cells or separate cell cultures that can be utilized in the method. According to the invention, the number of separate cell cultures can be two or more but more advantageously is at least 3, 4, 5, 6, 7, 8, 9, 10 or more separate cell cultures, for example, at least 12, at least 15, at least 20, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 48, at least 50, at least 75, at least 96, at least 100, at least 200, at least 300, at least 384, at least 400, at least 500, at least 1000, at least 10,000, at least 100,000, at least 500,000 or more.

A further advantageous property of the CFTR cells and cell lines of the invention is that they stably express CFTR in the absence of selective pressure. Selection pressure is applied in cell culture to select cells with desired sequences or traits, and is usually achieved by linking the expression of a polypeptide of interest with the expression of a selection marker that imparts to the cells resistance to a corresponding selective agent or pressure. Antibiotic selection includes, without limitation, the use of antibiotics (e.g., puromycin, neomycin, G418, hygromycin, bleomycin and the like). Non-antibiotic selection includes, without limitation, the use of nutrient deprivation, exposure to selective temperatures, exposure to mutagenic conditions and expression of fluorescent markers where the selection marker may be e.g., glutamine synthetase, dihydrofolate reductase (DHFR), oabain, thymidine kinase (TK), hypoxanthine guanine phosphororibosyltransferase (HGPRT) or a fluorescent protein such as GFP. Thus, in some embodiments, cells and cell lines of the invention are maintained in culture without any selective pressure. In further embodiments, cells and cell lines are maintained without any antibiotics. As used herein, cell maintenance refers to culturing cells after they have been selected as described above for their CFTR expression. Maintenance does not refer to the optional step of growing cells in a selective drug (e.g., an antibiotic) prior to cell sorting where drug resistance marker(s) introduced into the cells allow enrichment of stable transfectants in a mixed population.

Drug-free cell maintenance provides a number of advantages. For examples, drug-resistant cells do not always express the co-transfected transgene of interest at adequate levels, because the selection relies on survival of the cells that have taken up the drug resistant gene, with or without the transgene. Further, selective drugs are often mutagenic or otherwise interfere with the physiology of the cells, leading to skewed results in cell-based assays. For example, selective drugs may decrease susceptibility to apoptosis (Robinson et al., Biochemistry, 36(37):11169-11178 (1997)), increase DNA repair and drug metabolism (Deffie et al., Cancer Res. 48(13):3595-3602 (1988)), increase cellular pH (Thiebaut et al., J Histochem Cytochem. 38(5):685-690 (1990); Roepe et al., Biochemistry. 32(41):11042-11056 (1993); Simon et al., Proc Natl Acad Sci USA. 91(3):1128-1132 (1994)), decrease lysosomal and endosomal pH (Schindler et al., Biochemistry. 35(9):2811-2817 (1996); Altan et al., J Exp Med. 187(10):1583-1598 (1998)), decrease plasma membrane potential (Roepe et al., Biochemistry. 32(41):11042-11056 (1993)), increase plasma membrane conductance to chloride (Gill et al., Cell. 71(1):23-32 (1992)) and ATP (Abraham et al., Proc Natl Acad Sci USA. 90(1):312-316 (1993)), and increase rates of vesicle transport (Altan et al., Proc Natl Acad Sci USA. 96(8):4432-4437 (1999)). GFP, a commonly used non-antibiotic selective marker, may cause cell death in certain cell lines (Hanazono et al., Hum Gene Ther. 8(11):1313-1319 (1997)). Thus, the cells and cell lines of this invention allow screening assays that are free from any artifact caused by selective drugs or markers. In some preferred embodiments, the cells and cell lines of this invention are not cultured with selective drugs such as antibiotics before or after cell sorting, so that cells and cell lines with desired properties are isolated by sorting, even when not beginning with an enriched cell population.

In another aspect, the invention provides methods of using the cells and cell lines of the invention. The cells and cell lines of the invention may be used in any application for which functional CFTR or mutant CFTRs are needed. The cells and cell lines may be used, for example, but not limited to, in an in vitro cell-based assay or an in vivo assay where the cells are implanted in an animal (e.g., a non-human mammal) to, e.g., screen for CFTR (e.g., CFTR mutant) modulators; produce protein for crystallography and binding studies; and investigate compound selectivity and dosing, receptor/compound binding kinetic and stability, and effects of receptor expression on cellular physiology (e.g., electrophysiology, protein trafficking, protein folding, and protein regulation). The cells and cell lines of the invention also can be used in knock down studies to study the roles of mutant CFTRs.

Cells and cell lines expressing different forms of CFTR can be used separately or together to identify CFTR modulators, including those specific for a particular mutant CFTR and to obtain information about the activities of individual mutant CFTRs. The present cells and cell lines may be used to identify the roles of different forms of CFTR in different CFTR pathologies by correlating the identity of in vivo forms of mutant CFTR with the identify of known forms of CFTR based on their response to various modulators. This allows selection of disease- or tissue-specific CFTR modulators for highly targeted treatment of such CFTR-related pathologies.

Modulators include any substance or compound that alters an activity of a CFTR. Modulators help identifying the relevant mutant CFTRs implicated in CFTR pathologies (i.e., pathologies related to ion conductance through various CFTR channels), and selecting tissue specific compounds for the selective treatment of such pathologies or for the development of related compounds useful in those treatments. In other aspects, a modulator may change the ability of another modulator to affect the function of a CFTR. For example, a modulator of a mutant CFTR that is not activated by forskolin may render that form of CFTR susceptible to activation by forskolin.

Stable cell lines expressing a CFTR mutant and panels of such cell lines (see above) can be used to screen modulators (including agonists, antagonists, potentiators and inverse agonists), e.g., in high-throughput compatible assays. Modulators so identified can then be assayed against other CFTR alleles to identify specific modulators specific for given CFTR mutants.

In some embodiments, the present invention provides a method for generating an in-vitro-correlate (“IVC”) for an in vivo physiological property of interest. An IVC is generated by establishing the activity profile of a compound with an in vivo physiological property on different CFTR mutants, e.g., a profile of the effect of a compound on the physiological property of different CFTR mutants. This can be accomplished by using a panel of cells or cell lines as disclosed above. This activity profile is representative of the in vivo physiological property and thus is an IVC of a fingerprint for the physiological property. In some embodiments, the in-vitro-correlate is an in-vitro-correlate for a negative side effect of a drug. In other embodiments, the in-vitro-correlate is an in-vitro-correlate for a beneficial effect of a drug.

In some embodiments, the IVC can be used to predict or confirm one or more physiological properties of a compound of interest. The compound may be tested for its activity against different CFTR mutants and the resulting activity profile is compared to the activity profile of IVCs that are generated as described herein. The physiological property of the IVC with an activity profile most similar to the activity profile of a compound of interest is predicted to be and/or confirmed to be a physiological property of the compound of interest.

In some embodiments, an IVC is established by assaying the activities of a compound against different CFTR mutants, or combinations thereof. Similarly, to predict or confirm the physiological activity of a compound, the activities of the compound can be tested against different CFTR mutants.

In some embodiments, the methods of the invention can be used to determine and/or predict and/or confirm to what degree a particular physiological effect is caused by a compound of interest. In certain embodiments, the methods of the invention can be used to determine and/or predict and/or confirm the tissue specificity of a physiological effect of a compound of interest.

In more specific embodiments, the activity profile of a compound of interest is established by testing the activity of the compound in a plurality of in vitro assays using cell lines that are engineered to express different CFTR mutants (e.g., a panel of cells expressing different CFTR mutants). In some embodiments, testing of candidate drugs against a panel of CFTR mutants can be used to correlate specific targets to adverse or undesired side-effects or therapeutic efficacy observed in the clinic. This information may be used to select well defined targets in high-throughput screening or during development of drugs with maximal desired and minimal off-target activity.

In certain embodiments, the physiological parameter is measured using functional magnetic resonance imaging (“fMRI”). Other imaging methods can also be used, for example, computed tomography (CT); computed axial tomography (CAT) scanning; diffuse optical imaging (DOI); diffuse optical tomography (DOT); event-related optical signal (EROS); near infrared spectroscopy (NIRS); magnetic resonance imaging (MRI); magnetoencephalography (MEG); positron emission tomography (PET) and single photon emission computed tomography (SPECT).

In certain embodiments, if the IVC represents an effect of the compound on the central nervous system (“CNS”), an IVC may be established that correlates with an fMRI pattern in the CNS. IVCs may be generated that correlate with activity of compounds in various tests and models, including human and animal testing models. Human diseases and disorders are listed, e.g., in The Merck Manual, 18th Edition (Hardcover), Mark H. Beers (Author), Robert S. Porter (Editor), Thomas V. Jones (Editor). Mental diseases and disorders are listed, in e.g., Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) Fourth Edition (Text Revision), by American Psychiatric Association.

IVCs using CFTR can also be generated for the following properties: regulation, secretion, quality, clearance, production, viscosity, or thickness of mucous, water absorption, retention, balance, passing, or transport across epithelial tissues (especially of lung, kidney, vascular tissues, eye, gut, small intestine, large intestine); sensory or taste perception of compounds; neuronal firing or CNS activity in response to active compounds; pulmonary indications; gastrointestinal indications such as bowel cleansing, Irritable Bowel Syndrome (IBS), drug-induced (i.e. opioid) constipation, constipation/CIC of bedridden patients, acute infectious diarrhea, E. coli, cholera, viral gastroenteritis, rotavirus, modulation of malabsorption syndromes, pediatric diarrhea (viral, bacterial, protozoan), HIV, or short bowel syndrome; fertility indications such as sperm motility or sperm capacitation; female reproductive indications, cervical mucus/vaginal secretion viscosity (i.e. hostile cervical mucus); contraception, such as compounds that negatively affect sperm motility or cervical mucous quality relevant for sperm motility; dry mouth, dry eye, glaucoma, runny nose; or endocrine indications, i.e. pancreatic function in CF patients.

To identify a CFTR modulator, one can expose a novel cell or cell line of the invention to a test compound under conditions in which the CFTR would be expected to be functional and then detect a statistically significant change (e.g., p<0.05) in CFTR activity compared to a suitable control, e.g., cells that are not exposed to the test compound. Positive and/or negative controls using known agonists or antagonists and/or cells expressing different mutant CFTRs may also be used. In some embodiments, the CFTR activity to be detected and/or measured is membrane depolarization, change in membrane potential, or fluorescence resulting from such membrane changes. One of ordinary skill in the art would understand that various assay parameters may be optimized, e.g., signal to noise ratio.

In some embodiments, one or more cells or cell lines of the invention are exposed to a plurality of test compounds, for example, a library of test compounds. A library of test compounds can be screened using the cell lines of the invention to identify one or more modulators. The test compounds can be chemical moieties including small molecules, polypeptides, peptides, peptide mimetics, antibodies or antigen-binding portions thereof. In the case of antibodies, they may be non-human antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. The antibodies may be intact antibodies comprising a full complement of heavy and light chains or antigen-binding portions of any antibody, including antibody fragments (such as Fab, Fab′, F(ab′)₂, Fd, Fv, dAb, and the like), single chain antibodies (scFv), single domain antibodies, all or an antigen-binding portion of a heavy chain or light chain variable region.

In some embodiments, prior to exposure to a test compound, the cells or cell lines of the invention may be modified by pretreatment with, for example, enzymes, including mammalian or other animal enzymes, plant enzymes, bacterial enzymes, enzymes from lysed cells, protein modifying enzymes, lipid modifying enzymes, and enzymes in the oral cavity, gastrointestinal tract, stomach or saliva. Such enzymes can include, for example, kinases, proteases, phosphatases, glycosidases, oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and the like. Alternatively, the cells and cell lines may be exposed to the test compound first followed by treatment to identify compounds that alter the modification of the CFTR by the treatment.

In some embodiments, large compound collections are tested for CFTR modulating activity in a cell-based, functional, high-throughput screen (HTS), e.g., using a 96-well, 384-well, 1536-well or higher density well format. In some embodiments, a test compound or multiple test compounds including a library of test compounds may be screened using more than one cell or cell line of the invention. If multiple cells or cell lines, each expressing a different non-mutant CFTR or mutant CFTR are used, one can identify modulators that are effective on multiple forms of CFTR or alternatively, modulators that are specific for a particular mutant or non-mutant CFTR and that do not modulate other mutant CFTRs. In the case of a cell or cell line of the invention that expresses a human CFTR, one can expose the cells to a test compound to identify a compound that modulates CFTR activity (either increasing or decreasing) for use in the treatment of disease or condition characterized by undesired CFTR activity, or the decrease or absence of desired CFTR activity.

In certain embodiments, an assay for CFTR activity is performed using a cell or cell line expressing a CFTR mutant (see, e.g., Table 1 and Table 2), or a panel of mutants. In one embodiment, the panel also includes a cell or cell line that expresses wild type CFTR. In certain embodiments, a protein trafficking corrector is added to the assay. Such protein trafficking correctors include, but are not limited to: 1) Glycerol (see, e.g., Brown C R et al., Cell Stress and Chaperones (1996) v1(2):117-125); 2) DMSO (see, e.g., Brown C R et al., Cell Stress and Chaperones (1996) v1(2):117-125); 3) Deuterated water (D20) (see, e.g., Brown C R et al., Cell Stress and Chaperones (1996) v1(2):117-125); 4) Methylamines such as Trimethylamine Oxide (TMAO)(see, e.g., Brown C R et al., Cell Stress and Chaperones (1996) v1(2):117-125); 5) Adamantyl sulfogalactosyl ceramide (adaSGC)(see, e.g., Park H J et al., Chemistry and Biology (2009) v16: 461-470); 6) Vasoactive intestinal peptide (VIP)(see, e.g., Journal of Biological Chemistry (1999) v112: 887-894); 7) Sodium Phenyl Butyrate (S-PBA)(see, e.g., Singh O V et al., Molecular and Cellular Proteomics (2008) v7:1099-1110); 8) VRT-325 (see, e.g., Wang Y et al., Journal of Biological Chemistry (2007) v282(46): 33247-33257); 9) VRT-422 (see, e.g., Van Goor F et al., American Journal of Physiology Lung Cell Molecular Physiology (2006) v290: L1117-1130); 10) Corrector 2b (see, e.g., Wang Y et al., Journal of Biological Chemistry (2007) v282(46): 33247-33257); 11) Corrector 3a (see, e.g., Wang Y et al., Journal of Biological Chemistry (2007) v282(46): 33247-33257); 12) Corrector 4a (see, e.g., Wang Y et al., Journal of Biological Chemistry (2007) v282(46): 33247-33257); 13) Curcumin (see, e.g., Robert R et al., Molecular Pharmacology (2008) v73: 478-489); 14) Sildenafil analog (KM11060)(see, Robert R et al., e.g., Molecular Pharmacology (2008) v73: 478-489); 15) Alanine, Glutamic Acid, Proline, GABA, Taruine, Sucrose, Trehalose, Myo-inositol, Arabitol, Mannitol, Mannose, Sucrose, Betaine, Glycerophosphorylcholine, Sarcosine (see, e.g., Welch W J et al., Cell Stress and Chaperones (1996) v1(2): 109-115); and 16) N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide with the formula of

In certain embodiments, panels of cells or cell lines as described above can be used to test protein trafficking correctors. In certain embodiments, panels of cells or cell lines as described above can be used to screen for protein trafficking correctors.

In other embodiments, the assay of CFTR activity on a CFTR mutant is performed in the absence of a protein trafficking corrector. In some cases, the sensitivity of the CFTR activity assay is the same with or without the use of a protein trafficking Corrector.

These and other embodiments of the invention may be further illustrated in the following non-limiting Examples.

EXAMPLES

Example 1

Generating a Stable CFTR-Expressing Cell Line

Generating Expression Constructs

Plasmid expression vectors that allowed streamlined cloning were generated based on pCMV-SCRIPT (Stratagene) and contained various necessary components for transcription and translation of a gene of interest, including: CMV and SV40 eukaryotic promoters; SV40 and HSV-TK polyadenylation sequences; multiple cloning sites; Kozak sequences; and drug resistance cassettes (i.e., puromycin). Ampicillin or neomycin resistance cassettes can also be used to substitute puromycin. A tag sequence (SEQ ID NO: 8) was then inserted into the multiple cloning site of the plasmid. A cDNA cassette encoding a human CFTR was then subcloned into the multiple cloning site upstream of the tag sequence, using Asc1 and Pac1 restriction endonucleases.

Generating Cell Lines

Step 1: Transfection

CHO cells were transfected with a plasmid encoding a human CFTR (SEQ ID NO: 1) using standard techniques. (Examples of reagents that may be used to introduce nucleic acids into host cells include, but are not limited to, LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, OLIGOFECTAMINE™, TFX™ reagents, FUGENE® 6, DOTAP/DOPE, Metafectine or FECTURIN™.)

Although drug selection is optional to produce the cells or cell lines of this invention, we included one drug resistance marker in the plasmid (i.e., puromycin). The CFTR sequence was under the control of the CMV promoter. An untranslated sequence encoding a Target Sequence for detection by a signaling probe was also present along with the sequence encoding the drug resistance marker. The target sequence utilized was Target Sequence 2 (SEQ ID NO: 8), and in this example, the CFTR gene-containing vector comprised Target Sequence 2 (SEQ ID NO: 8).

Step 2: Selection

Transfected cells were grown for 2 days in Ham's F12-FBS media (Sigma Aldrich, St Louis, Mo.) without antibiotics, followed by 10 days in 12.5 μg/ml puromycin-containing Ham's F12-FBS media. The cells were then transferred to Ham's F12-FBS media without antibiotics for the remainder of the time, prior to the addition of the signaling probe.

Step 3: Cell Passaging

Following enrichment on antibiotic, cells were passaged 5-14 times in the absence of antibiotic selection to allow time for expression that was not stable over the selected period of time to subside.

Step 4: Exposure of Cells to Fluorogenic Probes

Cells were harvested and transfected with Signaling Probe 2 (SEQ ID NO: 9) using standard techniques. (Examples of reagents that may be used to introduce nucleic acids into host cells include, but are not limited to, LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, OLIGOFECTAMINE™, TFX™ reagents, FUGENE® 6, DOTAP/DOPE, Metafectine or FECTURIN™.) Signaling Probe 2 (SEQ ID NO: 9) bound Target Sequence 2 (SEQ ID NO: 8). The cells were then collected for analysis and sorted using a fluorescence activated cell sorter.

Target Sequence Detected by Signaling Probe

Target Sequence 2

	(SEQ ID NO: 8)
	5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′

Signaling Probe

Signaling Probe 2 (supplied as 100 μM stock)

(SEQ ID NO: 9)

5′-CY5.5 GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGC BHQ2-3′

BHQ2 in Signaling Probe 2 can be substituted with BHQ3 or a gold particle.

Target Sequence 2 and Signaling Probe 2 can be replaced by Target Sequence 1 and Signaling Probe 1, respectively.

Target Sequence 1

	(SEQ ID NO: 3)
	5′-GTTCTTAAGGCACAGGAACTGGGAC-3′

Signaling Probe 1 (supplied as 100 μM stock)

5′-Cy5 GCCAGTCCCAGTTCCTGTGCCTTAAGAACCTCGC BHQ2-3′

(SEQ ID NO: 6)

BHQ2 in Signaling Probe 1 can be substituted with BHQ3 or a gold particle.

In addition, a similar probe using a Quasar® Dye (BioSearch) with spectral properties similar to Cy5 is used in certain experiments against Target Sequence 1.

In some experiments, 5-MedC and 2-amino dA mixmers are used rather than DNA probes.

A non-targeting FAM labeled probe is also used as a loading control.

Step 5: Isolation of Positive Cells

The cells were dissociated and collected for analysis and sorting using a fluorescence activated cell sorter (Beckman Coulter, Miami, Fla.). Standard analytical methods were used to gate cells fluorescing above background and to isolate individual cells falling within the gate into bar-coded 96-well plates. The following gating hierarchy was used:

coincidence gate→singlets gate→live gate→Sort gate in plot FAM vs. Cy5.5: 0.1-0.4% of cells according to standard procedures in the field.
Step 6: Additional Cycles of Steps 1-5 and/or 3-5

Steps 1-5 and/or 3-5 were repeated to obtain a greater number of cells. Two rounds of steps 1-5 were performed, and for each of these rounds, two internal cycles of steps 3-5 were performed.

Step 7: Estimation of Growth Rates for the Populations of Cells

The plates were transferred to a Microlab Star (Hamilton Robotics). Cells were incubated for 9 days in 100 μl of 1:1 mix of fresh complete growth media and 2 to 3 day-conditioned growth media, supplemented with 100 units/ml penicillin and 0.1 mg/ml streptomycin. Then the cells were dispersed by trypsinization once or twice to minimize clumps and later transferred to new 96-well plates. Plates were imaged to determine confluency of wells (Genetix). Each plate was focused for reliable image acquisition across the plate. Reported confluencies of greater than 70% were not relied upon. Confluency measurements were obtained on consecutive days between days 1 and 10 post-dispersal and used to calculate growth rates.

Step 8: Binning Populations of Cells According to Growth Rate Estimates

Cells were binned (independently grouped and plated as a cohort) according to growth rate less than two weeks following the dispersal step in step 7. Each of the three growth bins was separated into individual 96 well plates; some growth bins resulted in more than one 96 well plate. Bins were calculated by considering the spread of growth rates and bracketing a high percentage of the total number of populations of cells. Bins were calculated to capture 12-16 hour differences in growth rate.

Cells can have doubling times from less than 1 day to more than 2 week. In order to process the most diverse clones that at the same time can be reasonably binned according to growth rate, it may be preferable to use 3-9 bins with a 0.25 to 0.7 day difference among the bins. One skilled in the art will appreciate that the tightness of the bins and number of bins can be adjusted for the particular situation and that the tightness and number of bins can be further adjusted if cells are synchronized for their cell cycle.

Step 9: Replica Plating to Speed Parallel Processing and Provide Stringent Quality Control

The plates were incubated under standardized and fixed conditions (i.e., Ham's F12-FBS media, 37° C./5% CO2) without antibiotics. The plates of cells were split to produce 4 sets of 96 well plates (3 sets for freezing, 1 set for assay and passage). Distinct and independent tissue culture reagents, incubators, personnel, and carbon dioxide sources were used for each of the sets of the plates. Quality control steps were taken to ensure the proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated hood with only that reagent in the hood while a second designated person monitored to avoid mistakes. Conditions for liquid handling were set to eliminate cross contamination across wells. Fresh tips were used for all steps or stringent tip washing protocols were used. Liquid handling conditions were set for accurate volume transfer, efficient cell manipulation, washing cycles, pipetting speeds and locations, number of pipetting cycles for cell dispersal, and relative position of tip to plate.

Step 10: Freezing Early Passage Stocks of Populations of Cells

Three sets of plates were frozen at −70 to −80° C. Plates in the set were first allowed to attain confluencies of 70 to 100%. Medium was aspirated and 90% FBS and 10% DMSO was added. The plates were individually sealed with Parafilm, individually surrounded by 1 to 5 cm of foam, and then placed into a −80° C. freezer.

Step 11: Methods and Conditions for Initial Transformative Steps to Produce Viable, Stable and Functional (VSF) Cell Lines

The remaining set of plates was maintained as described in step 9. All cell splitting was performed using automated liquid handling steps, including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersal steps.

Step 12: Normalization Methods to Correct any Remaining Variability of Growth Rates

The consistency and standardization of cell and culture conditions for all populations of cells was controlled. Differences across plates due to slight differences in growth rates were controlled by normalization of cell numbers across plates and occurred every 8 passages after the rearray. Populations of cells that were outliers were detected and eliminated.

Step 13: Characterization of Population of Cells

The cells were maintained for 6 to 10 weeks post rearray in culture. During this time, we observed size, morphology, tendency towards microconfluency, fragility, response to trypsinization and average circularity post-trypsinization, or other aspects of cell maintenance such as adherence to culture plate surfaces and resistance to blow-off upon fluid addition as part of routine internal quality control to identify robust cells. Such benchmarked cells were then admitted for functional assessment.

Step 14: Assessment of Potential Functionality of Populations of Cells Under VSF Conditions

Populations of cells were tested using functional criteria. Membrane potential dye kits (Molecular Devices, MDS) were used according to manufacturer's instructions.

Cells were tested at varying densities in 384-well plates (i.e., 12.5×10³ to 20×10³ cells/per well) and responses were analyzed. Time between cell plating and assay read was tested. Dye concentration was also tested. Dose response curves and Z′ scores were both calculated as part of the assessment of potential functionality.

The following steps (i.e., steps 15-18) can also be conducted to select final and back-up viable, stable, and functional cell lines.

Step 15:

The functional responses from experiments performed at low and higher passage numbers are compared to identify cells with the most consistent responses over defined periods of time (e.g., 3-9 weeks). Other characteristics of the cells that change over time are also noted.

Step 16:

Populations of cells meeting functional and other criteria are further evaluated to determine those most amenable to production of viable, stable and functional cell lines. Selected populations of cells are expanded in larger tissue culture vessels and the characterization steps described above are continued or repeated under these conditions. At this point, additional standardization steps, such as different cell densities; time of plating, length of cell culture passage; cell culture dishes format and coating; fluidics optimization, including speed and shear force; time of passage; and washing steps, are introduced for consistent and reliable passages.

In addition, viability of cells at each passage is determined. Manual intervention is increased and cells are more closely observed and monitored. This information is used to help identify and select final cell lines that retain the desired properties. Final cell lines and back-up cell lines are selected that show appropriate adherence/stickiness, growth rate, and even plating (lack of microconfluency) when produced following this process and under these conditions.

Step 17: Establishment of Cell Banks

The low passage frozen stocks corresponding to the final cell line and back-up cell lines are thawed at 37° C., washed two times with Ham's F12-FBS and then incubated in Ham's F12-FBS. The cells are then expanded for a period of 2 to 4 weeks. Cell banks of clones for each final and back-up cell line are established, with 25 vials for each clonal cells being cryopreserved.

Step 18:

At least one vial from the cell bank is thawed and expanded in culture. The resulting cells are tested to determine if they meet the same characteristics for which they are originally selected.

Example 2

Characterizing Stable Cell Lines for Native CFTR Function

We used a high-throughput compatible fluorescence membrane potential assay to characterize native CFTR function in the produced stable CFTR-expressing cell lines.

CHO cell lines stably expressing CFTR were maintained under standard cell culture conditions in Ham's F12 medium supplemented with 10% fetal bovine serum and glutamine. On the day before assay, the cells were harvested from stock plates and plated into black clear-bottom 384 well assay plates at a density that is sufficient to attain 90% confluency on the day of the assay. The assay plates were maintained in a 37° C. cell culture incubator under 5% CO₂ for 22-24 hours. The media was then removed from the assay plates and blue membrane potential dye (Molecular Devices Inc.) diluted in loading buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) was added and allowed to incubate for 1 hour at 37° C. The assay plates were then loaded on a fluorescent plate reader (Hamamatsu FDSS) and a cocktail of forskolin and IBMX dissolved in compound buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) was added.

Representative data from the fluorescence membrane potential assay is presented in FIGS. 1A and 1B. The ion flux attributable to functional CFTR in stable CFTR-expressing CHO cell lines (cell line 1, M11, J5, E15, and 015) were all higher than control cells lacking CFTR as indicated by the assay response.

The ion flux attributable to functional CFTR in stable CFTR-expressing CHO cell lines (cell line 1, M11, J5, E15, and 015) were also all higher than transiently CFTR-transfected CHO cells (FIGS. 1A and 1B). The transiently CFTR-transfected cells were generated by plating CHO cells at 5-16 million per 10 cm tissue culture dish and incubating them for 18-20 hours before transfection. A transfection complex consisting of lipid transfection reagent and plasmids encoding CFTR was directly added to each dish. The cells were then incubated at 37° C. in a CO2 incubator for 6-12 hours. After incubation, the cells were lifted, plated into black clear-bottom 384 well assay plates, and assayed for function using the above-described fluorescence membrane potential assay.

For forskolin dose-response experiments, cells of the produced stable CFTR-expressing cell lines, plated at a density of 15,000 cells/well in a 384-well plate were challenged with increasing concentration of forskolin, a known CFTR agonist. The cellular response as a function of changes in cell fluorescence was monitored over time by a fluorescent plate reader (Hamamatsu FDSS). Data were then plotted as a function of forskolin concentration and analyzed using non-linear regression analysis using GraphPad Prism 5.0 software, resulting in an EC₅₀ of 256 nM (FIG. 2). The produced CFTR-expressing cell line shows a EC₅₀ value of forskolin within the ranges of EC₅₀ of forskolin previously reported in other cell lines (between 250 and 500 nM) (Galietta et al., Am J Physiol Cell Physiol. 281(5): C1734-1742 (2001)), indicating the potency of the clone.

Example 3

Determination of Z′ Value for CFTR Cell-Based Assay

Z′ value for the produced stable CFTR-expressing cell line was calculated using a high-throughput compatible fluorescence membrane potential assay. The fluorescence membrane potential assay protocol was performed substantially according to the protocol in Example 2. Specifically for the Z′ assay, 24 positive control wells in a 384-well assay plate (plated at a density of 15,000 cells/well) were challenged with a CFTR activating cocktail of forskolin and IBMX. An equal number of wells were challenged with vehicle alone and containing DMSO (in the absence of activators). Cell responses in the two conditions were monitored using a fluorescent plate reader (Hamamatsu FDSS). Mean and standard deviations in the two conditions were calculated and Z′ was computed using the method disclosed in Zhang et al., J Biomol Screen, 4(2): 67-73, (1999). The Z′ value of the produced stable CFTR-expressing cell line was determined to be higher than or equal to 0.82.

Example 4

High-Throughput Screening and Identification of CFTR Modulators

A high-throughput compatible fluorescence membrane potential assay is used to screen and identify CFTR modulator. On the day before assay, the cells are harvested from stock plates into growth media without antibiotics and plated into black clear-bottom 384 well assay plates. The assay plates are maintained in a 37° C. cell culture incubator under 5% CO₂ for 19-24 hours. The media is then removed from the assay plates and blue membrane potential dye (Molecular Devices Inc.) diluted in load buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) is added and the cells are incubated for 1 hr at 37° C. Test compounds are solubilized in dimethylsulfoxide, diluted in assay buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) and then loaded into 384 well polypropylene micro-titer plates. The cell and compound plates are loaded into a fluorescent plate reader (Hamamatsu FDSS) and run for 3 minutes to identify test compound activity. The instrument adds a forskolin solution at a concentration of 300 nM-1 μM to the cells to allow either modulator or blocker activity of the previously added compounds to be observed. The activity of the compound is determined by measuring the change in fluorescence produced following the addition of the test compounds to the cells and/or following the subsequent agonist addition.

Example 5

Characterizing Stable CFTR-Expressing Cell Lines for Native CFTR Function Using Short-Circuit Current Measurements

Ussing chamber experiments are performed 7-14 days after plating CFTR-expressing cells (primary or immortalized epithelial cells including but not limited to lung and intestinal) on culture inserts (Snapwell, Corning Life Sciences). Cells on culture inserts are rinsed, mounted in an Ussing type apparatus (EasyMount Chamber System, Physiologic Instruments) and bathed with continuously gassed Ringer solution (5% CO₂ in O₂, pH 7.4) maintained at 37° C. containing 120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM glucose. The hemichambers are connected to a multichannel voltage and current clamp (VCC-MC8 Physiologic Instruments). Electrodes [agar bridged (4% in 1 M KCl) Ag—AgCl] are used and the inserts are voltage clamped to 0 mV. Transepithelial current, voltage and resistance are measured every 10 seconds for the duration of the experiment. Membranes with a resistance of <200 mΩs are discarded.

Example 6

Characterizing Stable CFTR-Expressing Cell Lines for Native CFTR Function Using Electrophysiological Assay

While both manual and automated electrophysiology assays have been developed and both can be applied to assay the native CFTR function, described below is the protocol for manual patch clamp experiments.

Cells are seeded at low densities and are used 2-4 days after plating. Borosilicate glass pipettes are fire-polished to obtain tip resistances of 2-4 mega Ω. Currents are sampled and low pass filtered. The extracellular (bath) solution contains: 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 10 mM mannitol, and 10 mM TES, pH 7.4. The pipette solution contains: 120 mM CsCl, 1 mM MgCl₂, 10 mM TEA-Cl, 0.5 mM EGTA, 1 mM Mg-ATP, and 10 mM HEPES (pH 7.3). Membrane conductances are monitored by alternating the membrane potential between −80 mV and −100 mV. Current-voltage relationships are generated by applying voltage pulses between −100 mV and +100 mV in 20-mV steps.

Example 7

Generating a Stable CFTR-ΔF508 Expressing Cell Line

Generating Expression Constructs

Plasmid expression vectors that allowed streamlined cloning were generated based on pCMV-SCRIPT (Stratagene) and contained various necessary components for transcription and translation of a gene of interest, including: CMV and SV40 eukaryotic promoters; SV40 and HSV-TK polyadenylation sequences; multiple cloning sites; Kozak sequences; and drug resistance cassettes (i.e., puromycin). Ampicillin or neomycin resistance cassettes can also be used to substitute puromycin. A tag sequence (SEQ ID NO: 8) was then inserted into the multiple cloning site of the plasmid. A cDNA cassette encoding a human CFTR was then subcloned into the multiple cloning site upstream of the tag sequence, using Asc1 and Pac1 restriction endonucleases. A site-directed mutagenesis (Stratagene) was then used to delete a single phenylalanine amino-acid at position 508 to generate plasmid encoding human CFTR-ΔF508 (SEQ ID NO: 7). The above-described mutagenesis method is compatible with high-throughput generation of any number of various CFTR alleles (either currently known or as may become known in the future).

Generating Cell Lines

Step 1: Transfection

CHO cells were transfected with a plasmid encoding a human CFTR-ΔF508 (SEQ ID NO: 7) using standard techniques. (Examples of reagents that may be used to introduce nucleic acids into host cells include, but are not limited to, LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, OLIGOFECTAMINE™, TFX™ reagents, FUGENE® 6, DOTAP/DOPE, Metafectine or FECTURIN™.)

Although drug selection is optional to produce the cells or cell lines of this invention, we included one drug resistance marker in the plasmid (i.e., puromycin). The CFTR-ΔF508 sequence was under the control of the CMV promoter. An untranslated sequence encoding a Target Sequence for detection by a signaling probe was also present along with the sequence encoding the drug resistance marker. The target sequence utilized was Target Sequence 2 (SEQ ID NO: 8), and in this example, the CFTR-ΔF508-containing vector comprised Target Sequence 2 (SEQ ID NO: 8).

Step 2: Selection

Transfected cells were grown for 2 days in Ham's F12-FBS media (Sigma Aldrich, St. Louis, Mo.) without antibiotics, followed by 10 days in 12.5 μg/ml puromycin-containing Ham's F12-FBS media. The cells were then transferred to Ham's F12-FBS media without antibiotics for the remainder of the time, prior to the addition of the signaling probe.

Step 3: Cell Passaging

Following enrichment on antibiotic, cells were passaged 5-14 times in the absence of antibiotic selection to allow time for expression that was not stable over the selected period of time to subside.

Step 4: Exposure of Cells to Fluorogenic Probes

Cells were harvested and transfected with Signaling Probe 2 (SEQ ID NO: 9) using standard techniques. (Examples of reagents that may be used to introduce nucleic acids into host cells include, but are not limited to, LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, OLIGOFECTAMINE™, TFX™ reagents, FUGENE® 6, DOTAP/DOPE, Metafectine or FECTURIN™.) Signaling Probe 2 (SEQ ID NO: 9) bound Target Sequence 2 (SEQ ID NO: 8). The cells were then collected for analysis and sorted using a fluorescence activated cell sorter.

Target Sequence Detected by Signaling Probe

Target Sequence 2

	(SEQ ID NO: 8)
	5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′

Signaling Probe

Signaling Probe 2 (supplied as 100 μM stock)

(SEQ ID NO: 9)

5′-CY5.5 GCGAGTCGCAGAACGACAGGGTTAACTTCCTCGC BHQ2-3′

BHQ2 in Signaling Probe 2 can be substituted with BHQ3 or a gold particle.

Target Sequence 2 and Signaling Probe 2 can be replaced by Target Sequence 1 and Signaling Probe 1, respectively.

Target Sequence 1

	(SEQ ID NO: 3)
	5′-GTTCTTAAGGCACAGGAACTGGGAC-3′

Signaling Probe 1 (supplied as 100 μM stock)

5′-Cy5 GCCAGTCCCAGTTCCTGTGCCTTAAGAACCTCGC BHQ2-3′

(SEQ ID NO: 6)

BHQ2 in Signaling Probe 1 can be substituted with BHQ3 or a gold particle.

In addition, a similar probe using a Quasar® Dye (BioSearch) with spectral properties similar to Cy5 is used in certain experiments against Target Sequence 1.

In some experiments, 5-MedC and 2-amino dA mixmers are used rather than DNA probes.

A non-targeting FAM labeled probe is also used as a loading control.

Step 5: Isolation of Positive Cells

The cells were dissociated and collected for analysis and sorting using a fluorescence activated cell sorter (Beckman Coulter, Miami, Fla.). Standard analytical methods were used to gate cells fluorescing above background and to isolate individual cells falling within the gate into bar-coded 96-well plates. The following gating hierarchy was used:

coincidence gateΘsinglets gate→live gate→Sort gate in plot FAM vs. Cy5.5: 0.1-0.5% of cells according to standard procedures in the field.
Step 6: Additional Cycles of Steps 1-5 and/or 3-5

Steps 1-5 and/or 3-5 were repeated to obtain a greater number of cells. Two rounds of steps 1-5 were performed, and for each of these rounds, two internal cycles of steps 3-5 were performed.

Step 7: Estimation of Growth Rates for the Populations of Cells

The plates were transferred to a Microlab Star (Hamilton Robotics). Cells were incubated for 9 days in 100 μl of 1:1 mix of fresh complete growth media and 2 to 3 day-conditioned growth media, supplemented with 100 units/ml penicillin and 0.1 mg/ml streptomycin. Then the cells were dispersed by trypsinization once or twice to minimize clumps and later transferred to new 96-well plates. Plates were imaged to determine confluency of wells (Genetix). Each plate was focused for reliable image acquisition across the plate. Reported confluencies of greater than 70% were not relied upon. Confluency measurements were obtained on consecutive days between days 1 and 10 post-dispersal and used to calculate growth rates.

Step 8: Binning Populations of Cells According to Growth Rate Estimates

Cells were binned (independently grouped and plated as a cohort) according to growth rate less than two weeks following the dispersal step in step 7. Each of the three growth bins was separated into individual 96 well plates; some growth bins resulted in more than one 96 well plate. Bins were calculated by considering the spread of growth rates and bracketing a high percentage of the total number of populations of cells. Bins were calculated to capture 12-16 hour differences in growth rate.

Cells can have doubling times from less than 1 day to more than 2 week. In order to process the most diverse clones that at the same time can be reasonably binned according to growth rate, it may be preferable to use 3-9 bins with a 0.25 to 0.7 day doubling time per bin. One skilled in the art will appreciate that the tightness of the bins and number of bins can be adjusted for the particular situation and that the tightness and number of bins can be further adjusted if cells are synchronized for their cell cycle.

Step 9: Replica Plating to Speed Parallel Processing and Provide Stringent Quality Control

The plates were incubated under standardized and fixed conditions (i.e., Ham's F12-FBS media, 37° C./5% CO2) without antibiotics. The plates of cells were split to produce 2 sets of 96 well plates (1 set for freezing, 1 set for assay and passage). Distinct and independent tissue culture reagents, incubators, personnel, and carbon dioxide sources were used for each of the sets of the plates. Quality control steps were taken to ensure the proper production and quality of all tissue culture reagents: each component added to each bottle of media prepared for use was added by one designated person in one designated hood with only that reagent in the hood while a second designated person monitored to avoid mistakes. Conditions for liquid handling were set to eliminate cross contamination across wells. Fresh tips were used for all steps or stringent tip washing protocols were used. Liquid handling conditions were set for accurate volume transfer, efficient cell manipulation, washing cycles, pipetting speeds and locations, number of pipetting cycles for cell dispersal, and relative position of tip to plate.

Step 10: Freezing Early Passage Stocks of Populations of Cells

One set of plate was frozen at −70 to −80° C. Plates were first allowed to attain confluencies of 70 to 100%. Medium was aspirated and 90% FBS and 10% DMSO was added. The plates were individually sealed with Parafilm, individually surrounded by 1 to 5 cm of foam, and then placed into a −80° C. freezer.

Step 11: Methods and Conditions for Initial Transformative Steps to Produce Viable, Stable and Functional (VSF) Cell Lines

The remaining set of plates was maintained as described in step 9. All cell splitting was performed using automated liquid handling steps, including media removal, cell washing, trypsin addition and incubation, quenching and cell dispersal steps.

Step 12: Normalization Methods to Correct any Remaining Variability of Growth Rates

The consistency and standardization of cell and culture conditions for all populations of cells are controlled. Differences across plates due to slight differences in growth rates are controlled by normalization of cell numbers across plates and occurred every 8 passages after the re-array. Populations of cells that are outliers are detected and eliminated.

Step 13: Characterization of Population of Cells

The cells were maintained for 6 to 10 weeks post rearray in the culture. During this time, we observed size, morphology, tendency towards microconfluency, fragility, response to trypsinization and average circularity post-trypsinization, or other aspects of cell maintenance such as adherence to culture plate surfaces and resistance to blow-off upon fluid addition as part of routine internal quality control to identify robust cells. Such benchmarked cells were then admitted for functional assessment.

Step 14: Assessment of Potential Functionality of Populations of Cells Under VSF Conditions

Populations of cells were tested for receptor function using a high throughput compatible fluorescence based membrane potential dye kit (Molecular Devices, MDS) according to manufacturer's instructions.

Population of CHO cells stably expressing CFTR-ΔF508 were maintained under standard cell culture conditions in Ham's F12 medium supplemented with 10% fetal bovine serum and glutamine. On the day before assay, the cells were harvested from stock plates. The cells were plated into black clear-bottom 384 well assay plates at a density that was sufficient to attain 90% confluency on the day of the assay, with or without a protein trafficking corrector, Chembridge compound #5932794 (Chembridge, San Diego, Calif.) (Yoo et al., (2008) Bioorganic & Medicinal Chemistry Letters; 18(8): 2610-2614). This compound is N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide, and has the formula of

The assay plates were maintained in a 37° C. cell culture incubator under 5% CO₂ for 22-24 hours. The media was then removed from the assay plates and membrane potential dye diluted in loading buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) (blue or AnaSpec, Molecular Devices Inc.) was added, with or without a quencher of the membrane potential dye, and was allowed to incubate for 1 hour at 37° C. The quencher can be any quencher well known in the art, e.g., Dipicrylamine (DPA), Acid Violet 17 (AV17), Diazine Black (DB), HLB30818, Trypan Blue, Bromophenol Blue, HLB30701, HLB30702, HLB30703, Nitrazine Yellow, Nitro Red, DABCYL (Molecular Probes), QSY (Molecular Probes), metal ion quenchers (e.g., Co²⁺, Ni²⁺, Cu²⁺), and iodide ion.

The assay plates were then loaded on a fluorescent plate reader (Hamamatsu FDSS) and a cocktail of forskolin and IBMX dissolved in compound buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) was added.

Representative data from the fluorescence membrane potential assay are presented in FIGS. 3A-3F. The ion flux attributable to functional CFTR-ΔF508 in stable CFTR-ΔF508 expressing CHO cell lines were identified by comparing the receptor's response to forskolin (30 μM)+IBMX (100 μM) cocktail against DMSO+Buffer controls (FIGS. 3A-3F) either in the presence or absence of the protein trafficking corrector—Chembridge compound #5932794. FIGS. 3A and 3B show responding and non-responding (control) clones assayed using blue membrane potential dye in the presence of the protein trafficking corrector (15-25 μM); FIGS. 3C and 3D show responding and non-responding (control) clones assayed using AnaSpec membrane potential dye in the presence of the protein trafficking corrector (15-25 μM). FIGS. 3E and 3F show responding and non-responding (control) clones assayed using AnaSpec membrane potential dye in the absence of the protein trafficking corrector.

Cells will be tested at varying densities in 384-well plates (i.e., 12.5×10³ to 20×10³ cells/per well) and responses will be analyzed. Time between cell plating and assay read will be tested. Dye concentration will also be tested. Dose response curves and Z′ scores can be calculated as part of the assessment of potential functionality.

The following steps (i.e., steps 15-18) can also be conducted to select final and back-up viable, stable, and functional cell lines.

Step 15:

The functional responses from experiments performed at low and higher passage numbers are compared to identify cells with the most consistent responses over defined periods of time (e.g., 3-9 weeks). Other characteristics of the cells that change over time are also noted.

Step 16:

Populations of cells meeting functional and other criteria will be further evaluated to determine those most amenable to production of viable, stable and functional cell lines. Selected populations of cells will be expanded in larger tissue culture vessels and the characterization steps described above will be continued or repeated under these conditions. At this point, additional standardization steps, such as different cell densities; time of plating, length of cell culture passage; cell culture dishes format—(note: not explored); fluidics optimization, including speed and shear force; time of passage; and washing steps, will be introduced for consistent and reliable passages.

In addition, viability of cells at each passage will be determined. Manual intervention will be increased and cells will be more closely observed and monitored. This information is used to help identify and select final cell lines that retain the desired properties. Final cell lines and back-up cell lines will be selected that show appropriate adherence/stickiness, growth rate, and even plating (lack of microconfluency) when produced following this process and under these conditions.

Step 17: Establishment of Cell Banks

The low passage frozen stocks corresponding to the final cell line and back-up cell lines will be thawed at 37° C., washed once with Ham's F12-FBS and then incubated in Ham's F12-FBS. The cells will be then expanded for a period of 2 to 4 weeks. Cell banks of clones for each final and back-up cell line will be established, with 25 vials for each clonal cells being cryopreserved.

Step 18:

At least one vial from the cell bank will be thawed and expanded in culture. The resulting cells will be tested to determine if they meet the same characteristics for which they are originally selected.

Example 8

Characterizing Stable Cell Lines for CFTR-ΔF508 Function

We will use a high-throughput compatible fluorescence membrane potential assay to characterize CFTR-ΔF508 function in the produced stable CFTR-ΔF508 expressing cell lines.

CHO cell lines stably expressing CFTR-ΔF508 will be maintained under standard cell culture conditions in Ham's F12 medium supplemented with 10% fetal bovine serum and glutamine. On the day before assay, the cells will be harvested from stock plates and plated into black clear-bottom 384 well assay plates in the presence or absence of a protein trafficking corrector (e.g., Chembridge compound #5932794, N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide). The assay plates will be maintained in a 37° C. cell culture incubator under 5% CO₂ for 22-24 hours. The media will be then removed from the assay plates and blue membrane potential dye (Molecular Devices Inc.) diluted in loading buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) will be added and allowed to incubate for 1 hour at 37° C. The assay plates will be then loaded on a fluorescent plate reader (Hamamatsu FDSS) and a cocktail of forskolin and IBMX dissolved in compound buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) will be added. Stable cell-lines expressing CFTR-ΔF508 protein will be identified by measuring the change in fluorescence produced following the addition of the agonist cocktail.

Stable cell lines expressing the CFTR-ΔF508 protein will be then characterized with increasing doses of forskolin. For forskolin dose-response experiments, cells of the produced stable CFTR-ΔF508 expressing cell lines, plated at a density of 15,000 cells/well in a 384-well plate will be challenged with increasing concentrations of forskolin, a CFTR agonist. The cellular response as a function of changes in cell fluorescence will be monitored over time by a fluorescent plate reader (Hamamatsu FDSS). Data will be then plotted as a function of forskolin concentration and analyzed using non-linear regression analysis using GraphPad Prism 5.0 software to determine the EC50 value.

Example 9

Determination of Z′ Value for CFTR-ΔF508 Cell-Based Assay

Z′ value for the produced stable CFTR-ΔF508 expressing cell line will be calculated using a high-throughput compatible fluorescence membrane potential assay. The fluorescence membrane potential assay protocol will be performed substantially according to the protocol in Example 8. Specifically for the Z′ assay, 24 positive control wells in a 384-well assay plate (plated at a density of 15,000 cells/well) will be challenged with a CFTR activating cocktail of forskolin and IBMX. An equal number of wells will be challenged with vehicle alone and containing DMSO (in the absence of activators). The assay can be performed in the presence or absence of a protein trafficking corrector (e.g., Chembridge compound #5932794, N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide). Cell responses in the two conditions will be monitored using a fluorescent plate reader (Hamamatsu FDSS). Mean and standard deviations in the two conditions will be calculated and Z′ was computed using the method disclosed in Zhang et al., J Biomol Screen, 4(2): 67-73 (1999).

Example 10

High-Throughput Screening and Identification of CFTR-ΔF508 Modulators

A high-throughput compatible fluorescence membrane potential assay will be used to screen and identify CFTR-ΔF508 modulator(s). Modulating compounds may either enhance protein trafficking to the cell surface or modulate CFTR-ΔF508 agonists (for example, Forskolin) by increasing or decreasing the agonist activity. On the day before assay, the cells will be harvested from stock plates into growth media without antibiotics and plated into black clear-bottom 384 well assay plates in the presence or absence of a protein trafficking corrector (e.g., Chembridge compound #5932794-N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide). The assay plates will be maintained in a 37° C. cell culture incubator under 5% CO₂ for 19-24 hours. The media will be then removed from the assay plates and blue membrane potential dye (Molecular Devices Inc.) diluted in load buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) will be added and the cells will be incubated for 1 hr at 37° C. Test compounds will be solubilized in dimethylsulfoxide, diluted in assay buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) and then loaded into 384 well polypropylene micro-titer plates. The cell and compound plates will be loaded into a fluorescent plate reader (Hamamatsu FDSS) and run for 3 minutes to identify test compound activity. The instrument will then add a forskolin solution at a concentration of 300 nM-1 μM to the cells to allow either modulator or blocker activity of the previously added compounds to be observed. The activity of the compound will be determined by measuring the change in fluorescence produced following the addition of the test compounds to the cells and/or following the subsequent agonist addition.

For identification of compounds that may promote cell surface trafficking of the CFTR-ΔF508 protein on the day before assay, the cells will be harvested from stock plates into growth media without antibiotics and plated into black clear-bottom 384 well assay plates in the presence of the test compounds for a period of 24 hours. Some wells in the 384 well plate will not receive any test compound as negative controls, while others wells in the 384 well plates will receive a protein trafficking corrector (e.g., Chembridge compound #5932794, N-{2-[(2-methoxyphenyl)amino]-4′-methyl-4,5′-bi-1,3-thiazol-2′-yl}benzamide hydrobromide) and serve as positive controls. The assay plates will be maintained in a 37° C. cell culture incubator under 5% CO₂ for 19-24 hours. The media will be then removed from the assay plates and blue membrane potential dye (Molecular Devices Inc.) diluted in load buffer (137 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) will be added and the cells will be incubated for 1 hr at 37° C. The assay plates will be then loaded on a fluorescent plate reader (Hamamatsu FDSS) and a cocktail of forskolin and IBMX dissolved in compound buffer (137 mM sodium gluconate, 5 mM potassium gluconate, 1.25 mM CaCl₂, 25 mM HEPES, 10 mM glucose) will be added. The activity of the test compounds will be determined by measuring the change in fluorescence produced following the addition of the agonist cocktail (i.e. forskolin+IBMX).

Example 11

Characterizing Stable CFTR-ΔF508 Expressing Cell Lines for CFTR-ΔF508 Function Using Short-Circuit Current Measurements

Ussing chamber experiments will be performed 7-14 days after plating CFTR-ΔF508 expressing cells (e.g., primary or immortalized epithelial cells including but not limited to lung and intestinal cells) on culture inserts (Snapwell, Corning Life Sciences). Cells on culture inserts will be rinsed, mounted in an Ussing type apparatus (EasyMount Chamber System, Physiologic Instruments) and bathed with continuously gassed Ringer solution (5% CO₂ in O₂, pH 7.4) maintained at 37° C. containing 120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 10 mM glucose. The hemichambers will be connected to a multichannel voltage and current clamp (VCC-MC8 Physiologic Instruments). Electrodes [agar bridged (4% in 1 M KCl) Ag—AgCl] will be used and the inserts will be voltage clamped to 0 mV. Transepithelial current, voltage, and resistance will be measured every 10 seconds for the duration of the experiment. Membranes with a resistance of <200 mils will be discarded.

Example 12

Characterizing Stable CFTR-ΔF508 Expressing Cell Lines for CFTR-ΔF508 Function Using Electrophysiological Assay

While both manual and automated electrophysiology assays have been developed and both can be applied to characterize stable CFTR-ΔF508 expressing cell lines for CFTR-ΔF508 function, described below is the protocol for manual patch clamp experiments.

Cells are seeded at low densities and are used 2-4 days after plating. Borosilicate glass pipettes are fire-polished to obtain tip resistances of 2-4 mega Ω. Currents will be sampled and low pass filtered. The extracellular (bath) solution will contain: 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 10 mM mannitol, and 10 mM TES, pH 7.4. The pipette solution will contain: 120 mM CsCl, 1 mM MgCl₂, 10 mM TEA-C1, 0.5 mM EGTA, 1 mM Mg-ATP, and 10 mM HEPES (pH 7.3). Membrane conductances will be monitored by alternating the membrane potential between −80 mV and −100 mV. Current-voltage relationships will be generated by applying voltage pulses between −100 mV and +100 mV in 20-mV steps.

LISTING OF SEQUENCES

Homo sapiens (H.s.) cystic fibrosis

transmembrane conductance regulator (CFTR)

nucleotide sequence (SEQ ID NO: 1):

atgcagaggtcgcctctggaaaaggccagcgttgtctccaaactttt

tttcagctggaccagaccaattttgaggaaaggatacagacagcgcc

tggaattgtcagacatataccaaatcccttctgttgattctgctgac

aatctatctgaaaaattggaaagagaatgggatagagagctggcttc

aaagaaaaatcctaaactcattaatgcccttcggcgatgttttttct

ggagatttatgttctatggaatctttttatatttaggggaagtcacc

aaagcagtacagcctctatactgggaagaatcatagcttcctatgac

ccggataacaaggaggaacgctctatcgcgatttatctaggcatagg

cttatgccttctctttattgtgaggacactgctcctacacccagcca

tttttggccttcatcacattggaatgcagatgagaatagctatgttt

agtttgatttataagaagactttaaagctgtcaagccgtgttctaga

taaaataagtattggacaacttgttagtctcctttccaacaacctga

acaaatttgatgaaggacttgcattggcacatttcgtgtggatcgct

cctttgcaagtggcactcctcatggggctaatctgggagttgttaca

ggcgtctgccttctgtggacttggtttcctgatagtccttgcccttt

ttcaggctgggctagggagaatgatgatgaagtacagagatcagaga

gctgggaagatcagtgaaagacttgtgattacctcagaaatgattga

aaatatccaatctgttaaggcatactgctgggaagaagcaatggaaa

aaatgattgaaaacttaagacaaacagaactgaaactgactcggaag

gcagcctatgtgagatacttcaatagctcagccttcttcttctcagg

gttctttgtggtgtttttatctgtgcttccctatgcactaatcaaag

gaatcatcctccggaaaatattcaccaccatctcattctgcattgtt

ctgcgcatggcggtcactcggcaatttccctgggctgtacaaacatg

gtatgactctatggagcaataaacaaaatacaggatttcttacaaaa

gcaagaatataagacattggaatataacttaacgactacagaagtag

tgatggagaatgtaacagcatctgggaggagggatttggggaattat

ttgagaaagcaaaacaaaacaataacaatagaaaaacttctaatggt

gatgacagcctcttcttcagtaatttctcacttcttggtactcctgt

cctgaaagatattaatttcaagatagaaagaggacagttgttggcgg

ttgctggatccactggagcaggcaagacttcacttctaatggtgatt

atgggagaactggagccttcagagggtaaaattaagcacagtggaag

aatttcattctgttctcagttttcctggattatgcctggcaccatta

aagaaaatatcatctttggtgtttcctatgatgaatatagatacaga

agcgtcatcaaagcatgccaactagaagaggacatctccaagtttgc

agagaaagacaatatagttcttggagaaggtggaatcacactgagtg

gaggtcaacgagcaagaatttctttagcaagagcagtatacaaagat

gctgatttgtatttattagactctcatttggatacctagatgtttta

acagaaaaagaaatatttgaaagctgtgtctgtaaactgatggctaa

caaaactaggattttggtcacttctaaaatggaacatttaaagaaag

ctgacaaaatattaattttgcatgaaggtagcagctatttttatggg

acattttcagaactccaaaatctacagccagactttagctcaaaact

catgggatgtgattctttcgaccaatttagtgcagaaagaagaaatt

caatcctaactgagaccttacaccgtttctcattagaaggagatgct

cctgtctcctggacagaaacaaaaaaacaatcttttaaacagactgg

agagtttggggaaaaaaggaagaattctattctcaatccaatcaact

ctatacgaaaattttccattgtgcaaaagactcccttacaaatgaat

ggcatcgaagaggattctgatgagcctttagagagaaggctgtcctt

agtaccagattctgagcagggagaggcgatactgcctcgcatcagcg

tgatcagcactggccccacgcttcaggcacgaaggaggcagtctgtc

ctgaacctgatgacacactcagttaaccaaggtcagaacattcaccg

aaagacaacagcatccacacgaaaagtgtcactggcccctcaggcaa

acttgactgaactggatatatattcaagaaggttatctcaagaaact

ggcttggaaataagtgaagaaattaacgaagaagacttaaaggagtg

cttttttgatgatatggagagcataccagcagtgactacatggaaca

cataccttcgatatattactgtccacaagagcttaatttttgtgcta

atttggtgcttagtaatttttctggcagaggtggctgcttctttggt

tgtgctgtggctccttggaaacactcctcttcaagacaaagggaata

gtactcatagtagaaataacagctatgcagtgattatcaccagcacc

agttcgtattatgtgttttacatttacgtgggagtagccgacacttt

gcttgctatgggattcttcagaggtctaccactggtgcatactctaa

tcacagtgtcgaaaattttacaccacaaaatgttacattctgttctt

caagcacctatgtcaaccctcaacacgttgaaagcaggtgggattct

taatagattctccaaagatatagcaattttggatgaccttctgcctc

ttaccatatttgacttcatccagttgttattaattgtgattggagct

atagcagttgtcgcagttttacaaccctacatctttgttgcaacagt

gccagtgatagtggcttttattatgttgagagcatatttcctccaaa

cctcacagcaactcaaacaactggaatctgaaggcaggagtccaatt

ttcactcatcttgttacaagcttaaaaggactatggacacttcgtgc

cttcggacggcagccttactttgaaactctgttccacaaagctctga

atttacatactgccaactggttatgtacctgtcaacactgcgctggt

tccaaatgagaatagaaatgatttttgtcatatcttcattgctgtta

ccttcatttccattttaacaacaggagaaggagaaggaagagttggt

attatcctgactttagccatgaatatcatgagtacattgcagtgggc

tgtaaactccagcatagatgtggatagcttgatgcgatctgtgagcc

gagtctttaagttcattgacatgccaacagaaggtaaacctaccaag

tcaaccaaaccatacaagaatggccaactctcgaaagttatgattat

tgagaattcacacgtgaagaaagatgacatctggccctcagggggcc

aaatgactgtcaaagatctcacagcaaaatacacagaaggtggaaat

gccatattagagaacatttccttctcaataagtectggccagagggt

gggcctcttgggaagaactggatcagggaagagtactttgttatcag

cttttttgagactactgaacactgaaggagaaatccagatcgatggt

gtgtcttgggattcaataactttgcaacagtggaggaaagcctttgg

agtgataccacagaaagtatttattttttctggaacatttagaaaaa

acttggatccctatgaacagtggagtgatcaagaaatatggaaagtt

gcagatgaggttgggctcagatctgtgatagaacagtttcctgggaa

gcttgactttgtccttgtggatgggggctgtgtcctaagccatggcc

acaagcagttgatgtgcttggctagatctgttctcagtaaggcgaag

atcttgctgcttgatgaacccagtgctcatttggatccagtaacata

ccaaataattagaagaactctaaaacaagcatttgctgattgcacag

taattctctgtgaacacaggatagaagcaatgctggaatgccaacaa

tttttggtcatagaagagaacaaagtgcggcagtacgattccatcca

gaaactgctgaacgagaggagcctcttccggcaagccatcagcccct

ccgacagggtgaagctctttccccaccggaactcaagcaagtgcaag

tctaagccccagattgctgctctgaaagaggagacagaagaagaggt

gcaagatacaaggctttga

H.s. CFTR amino acid sequence (SEQ ID NO: 2):

MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSAD

NLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVT

KAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPA

IFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNL

NKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLAL

FQAGLGRIVIMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEA

MEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYAL

IKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDF

LQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNININR

KTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTS

LLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYD

EYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLAR

AVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKM

EHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFS

AERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSI

LNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAI

LPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVS

LAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPA

VTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPL

QDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLP

LVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAIL

DDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLR

AYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETL

FHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGE

GEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPT

EGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAK

YTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEG

EIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSD

QEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARS

VLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEA

MLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHR

NSSKCKSKPQIAALKEETEEEVQDTRL

Target Sequence 1 (SEQ ID NO: 3):

5′-GTTCTTAAGGCACAGGAACTGGGAC-3′

H.s. CFTR mutant (ΔF508) nucleotide sequence

(SEQ ID NO: 4):

atgcagaggtcgcctctggaaaaggccagcgttgtctccaaacttat

ttcagctggaccagaccaattttgaggaaaggatacagacagcgcct

ggaattgtcagacatataccaaatcccttctgttgattctgctgaca

atctatctgaaaaattggaaagagaatgggatagagagctggcttca

aagaaaaatcctaaactcattaatgccatcggcgatgttttttctgg

agatttatgttctatggaatctttttatatttaggggaagtcaccaa

agcagtacagcctctatactgggaagaatcatagettcctatgaccc

ggataacaaggaggaacgctctatcgcgatttatctaggcataggct

tatgccttctctttattgtgaggacactgctcctacacccagccatt

tttggccttcatcacattggaatgcagatgagaatagctatgtttag

tttgatttataagaagactttaaagctgtcaagccgtgttctagata

aaataagtattggacaacttgttagtctectttccaacaacctgaac

aaatttgatgaaggacttgcattggcacatttcgtgtggatcgctcc

tttgcaagtggcactectcatggggctaatctgggagttgttacagg

cgtctgccttctgtggacttggtttcctgatagtccttgcccttttt

caggctgggctagggagaatgatgatgaagtacagagatcagagagc

tgggaagatcagtgaaagacttgtgattacctcagaaatgattgaaa

atatccaatctgttaaggcatactgctgggaagaagcaatggaaaaa

atgattgaaaacttaagacaaacagaactgaaactgactcggaaggc

agcctatgtgagatacttcaatagctcagccttcttcttctcagggt

tctttgtggtgtttttatctgtgcttccctatgcactaatcaaagga

atcatcctccggaaaatattcaccaccatctcattctgcattgttct

gcgcatggeggtcactcggcaatttccctgggctgtacaaacatggt

atgactctcttggagcaataaacaaaatacaggatttcttacaaaag

caagaatataagacattggaatataacttaacgactacagaagtagt

gatggagaatgtaacagccttctgggaggagggatttggggaattat

ttgagaaagcaaaacaaaacaataacaatagaaaaacttctaatggt

gatgacagcctcttcttcagtaatttctcacttcttggtactcctgt

cctgaaagatattaatttcaagatagaaagaggacagttgttggcgg

ttgctggatccactggagcaggcaagacttcacttctaatggtgatt

atgggagaactggagccttcagagggtaaaattaagcacagtggaag

aatttcattctgttctcagttttcctggattatgcctggcaccatta

aagaaaatatcatcggtgtttcctatgatgaatatagatacagaagc

gtcatcaaagcatgccaactagaagaggacatctccaagtttgcaga

gaaagacaatatagttcttggagaaggtggaatcacactgagtggag

gtcaacgagcaagaatttattagcaagagcagtatacaaagatgctg

atttgtatttattagactctccttttggatacctagatgttttaaca

gaaaaagaaatatttgaaagctgtgtctgtaaactgatggctaacaa

aactaggattttggtcacttctaaaatggaacatttaaagaaagctg

acaaaatattaattttgcatgaaggtagcagctatttttatgggaca

ttttcagaactccaaaatctacagccagactttagctcaaaactcat

gggatgtgattctttcgaccaatttagtgcagaaagaagaaattcaa

tcctaactgagaccttacaccgatctcattagaaggagatgctcctg

tctcctggacagaaacaaaaaaacaatcttttaaacagactggagag

tttggggaaaaaaggaagaattctattctcaatccaatcaactctat

acgaaaattttccattgtgcaaaagactcccttacaaatgaatggca

tcgaagaggattctgatgagcctttagagagaaggctgtccttagta

ccagattctgagcagggagaggcgatactgcctcgcatcagcgtgat

cagcactggccccacgcttcaggcacgaaggaggcagtctgtcctga

acctgatgacacactcagttaaccaaggtcagaacattcaccgaaag

acaacagcatccacacgaaaagtgtcactggcccacaggcaaacttg

actgaactggatatatattcaagaaggttatctcaagaaactggett

ggaaataagtgaagaaattaacgaagaagacttaaaggagtgctttt

ttgatgatatggagagcataccagcagtgactacatggaacacatac

cttcgatatattactgtccacaagagcttaatttttgtgctaatttg

gtgcttagtaatttttctggcagaggtggctgcttctttggttgtgc

tgtggctccttggaaacactcctcttcaagacaaagggaatagtact

catagtagaaataacagctatgcagtgattatcaccagcaccagttc

gtattatgtgttttacatttacgtgggagtagccgacactttgcttg

ctatgggattcttcagaggtctaccactggtgcatactctaatcaca

gtgtcgaaaattttacaccacaaaatgttacattctgttcttcaagc

acctatgtcaaccctcaacacgttgaaagcaggtgggattcttaata

gattctccaaagatatagcaattttggatgaccttctgcctcttacc

atatttgacttcatccagttgttattaattgtgattggagctatagc

agttgtcgcagttttacaaccctacatctttgttgcaacagtgccag

tgatagtggcttttattatgttgagagcatatttcctccaaacctca

cagcaactcaaacaactggaatctgaaggcaggagtccaattttcac

tcatcttgttacaagataaaaggactatggacacttcgtgccttcgg

acggcagccttactttgaaactctgttccacaaagctctgaatttac

atactgccaactggttcttgtacctgtcaacactgcgctggttccaa

atgagaatagaaatgatttttgtcatcttcttcattgctgttacctt

catttccattttaacaacaggagaaggagaaggaagagttggtatta

tcctgactttagccatgaatatcatgagtacattgcagtgggctgta

aactccagcatagatgtggatagcttgatgcgatctgtgagccgagt

ctttaagttcattgacatgccaacagaaggtaaacctaccaagtcaa

ccaaaccatacaagaatggccaactctcgaaagttatgattattgag

aattcacacgtgaagaaagatgacatctggccctcagggggccaaat

gactgtcaaagatctcacagcaaaatacacagaaggtggaaatgcca

tattagagaacatttccttctcaataagtcctggccagagggtgggc

ctatgggaagaactggatcagggaagagtactttgttatcagctttt

ttgagactactgaacactgaaggagaaatccagatcgatggtgtgtc

ttgggattcaataactttgcaacagtggaggaaagcctttggagtga

taccacagaaagtatttattttttctggaacatttagaaaaaacttg

gatccctatgaacagtggagtgatcaagaaatatggaaagttgcaga

tgaggttgggctcagatctgtgatagaacagtttcctgggaagcttg

actttgtccttgtggatgggggctgtgtcctaagccatggccacaag

cagttgatgtgcttggctagatctgttctcagtaaggcgaagatctt

gctgcttgatgaacccagtgctcatttggatccagtaacataccaaa

taattagaagaactctaaaacaagcatttgctgattgcacagtaatt

ctctgtgaacacaggatagaagcaatgctggaatgccaacaattttt

ggtcatagaagagaacaaagtgcggcagtacgattccatccagaaac

tgctgaacgagaggagcctatccggcaagccatcagccectccgaca

gggtgaagctctttccccaccggaactcaagcaagtgcaagtctaag

ccccagattgctgctctgaaagaggagacagaagaagaggtgcaaga

tacaaggctttga

YFP mutant (meYFP- H148Q/I152L) nucleotide

sequence (SEQ ID NO: 5):

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcct

ggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccg

gcgagggcgagggcgatgccacctacggcaagctgaccctgaagttc

atctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgac

caccttcggctacggcctgcagtgcttcgcccgctaccccgaccaca

tgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtc

caggagcgcaccatcttcttcaaggacgacggcaactacaagacccg

cgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagc

tgaagggcatcgacttcaaggaggacggcaacatcctggggcacaag

ctggagtacaactacaacagccaaaacgtctatctcatggccgacaa

gcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcg

aggacggcagcgtgcagctcgccgaccactaccagcagaacaccccc

atcggcgacggccccgtgctgctgcccgacaaccactacctgagcta

ccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatgg

tcctgctggagttcgtgaccgccgccgggatcactctcggcatggac

gagctgtacaagtaa

Signaling Probe 1 (SEQ ID NO: 6):

5′-Cy5 GCCAGTCCCAGTTCCTGTGCCTTAAGAACCT

CGC BHQ2-3′

H.s. CFTR mutant (ΔF508) amino acid sequence

(SEQ ID NO: 7):

MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSAD

NLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEV

TKAVQPLLLGRILASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHP

AIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNN

LNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLA

LFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAM

EKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALI

KGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFL

QKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTS

NGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLM

VIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIGVSYDEYRY

RSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYK

DADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLK

KADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERR

NSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPI

NSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRI

SVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQ

ANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTW

NTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKG

NSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHT

LITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLL

PLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFL

QTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKA

LNLHTANWELYLSTLRWFQMRIEMIEVIFFIAVTFISILTTGEGEGR

VGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKP

TKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEG

GNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQI

DGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIW

KVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSK

AKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLEC

QQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSK

CKSKPQIAALKEETEEEVQDTRL

Target Sequence 2 (SEQ ID NO: 8)

5′-GAAGTTAACCCTGTCGTTCTGCGAC-3′

Signaling Probe 2 (SEQ ID NO: 9)

5′-CY5.5 GCGAGTCGCAGAACGACAGGGTTAACTTCCT

CGC BHQ2-3′